Application of bioactive peptide in preparation of medicine for treating intimal hyperplasia after vascular injury

Abstract

The invention relates to an application of a bioactive peptide in preparation of a medicine for treating intimal hyperplasia after vascular injury. The bioactive peptide is vasostatin-2. The invention finds that the bioactive peptide vasostatin-2 reduces neointimal growth caused by mouse vascular injury and inhibits proliferation of in-vitro VSMCs through an ACE2 / NR1D1 / Gas1 pathway, so that the vasostatin-2 becomes an effective strategy for treating vascular intimal hyperplasia diseases, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the field of vascular intimal hyperplasia, and particularly relates to the application of bioactive peptides in the preparation of drugs for treating intimal hyperplasia after vascular injury. Background Art

[0002] Intimal hyperplasia is a major feature of vascular remodeling and is closely associated with the pathological processes of a variety of vascular diseases, including restenosis after angioplasty, aortic aneurysm, atherosclerosis, and vascular calcification. Although the incidence of restenosis after angioplasty and the resulting need for repeated revascularization have decreased in recent years, vascular restenosis remains a pathological problem of public health significance given the millions of drug-eluting stents (DES) and drug-coated balloons (DCBs) used each year worldwide.

[0003] Vascular smooth muscle cells (VSMCs) are key components of the arterial wall tunica media and are usually in a quiescent, contractile state, playing a vital role in maintaining vascular integrity and elasticity and regulating vascular contractility / dilation. However, stress stimuli, including vascular injury and inflammation, induce VSMCs to switch from a contractile state to a secretory state, manifested by enhanced proliferation and migration, leading to intimal hyperplasia. Inhibiting VSMC proliferation has become an effective method to alleviate pathological intimal hyperplasia. Therefore, the development of new therapeutic strategies and the revelation of the molecular mechanisms of these therapies on injury-induced VSMC proliferation and phenotypic transformation have attracted widespread attention.

[0004] Vasostatin-2 is a bioactive peptide derived from chromogranin A. Chromogranin A is a member of the chromogranin / secretogranin family of acidic glycoproteins and is primarily synthesized and stored in neuroendocrine cells, particularly in chromaffin cells of the adrenal medulla and sympathetic nervous system. Upon stimulation, chromogranin A undergoes proteolysis, releasing various bioactive peptides, including vasostatin-2. Vasostatin-2 has attracted much attention due to its diverse vascular biological functions and potential role in a variety of cardiovascular diseases. Studies have shown that vasostatin-2 levels are reduced in atherosclerotic plaques and peripheral blood mononuclear cells (PBMCs) in patients with coronary artery disease (CAD), and reduced serum vasostatin-2 levels are significantly associated with the severity of CAD. Injection of vasostatin-2 prevents heart failure in rats with myocardial infarction by inhibiting monocyte / macrophage recruitment. In addition, studies have shown that serum vasostatin-2 levels are associated with collateral angiogenesis in patients with diabetes. However, the role of vasostatin-2 in injury-induced vascular remodeling remains unclear. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an application of a bioactive peptide in the preparation of a drug for treating intimal hyperplasia after vascular injury, to explore the correlation between serum vasostatin-2 levels and restenosis after coronary angioplasty, and the role of vasostatin-2 in inhibiting intimal hyperplasia, and to provide a new strategy for the treatment of vascular remodeling diseases with intimal hyperplasia as the pathological mechanism.

[0006] The invention provides application of a bioactive peptide in preparing a drug for treating intimal hyperplasia after vascular injury, wherein the bioactive peptide is vasostatin-2.

[0007] Preferably, the vasostatin-2 reduces neointimal growth caused by vascular injury in mice and inhibits the proliferation of VSMCs in vitro via the ACE2 / NR1D1 / Gas1 pathway.

[0008] Preferably, the drug further comprises a pharmaceutically acceptable carrier and / or excipient.

[0009] Preferably, the pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0010] The present invention also provides a pharmaceutical composition for treating intimal hyperplasia after vascular injury, wherein the pharmaceutical composition comprises a therapeutically effective amount of the vasostatin-2.

[0011] Preferably, the dosage form of the pharmaceutical composition includes at least one of tablets, pills, powders, solutions, suspensions, emulsions, and granules.

[0012] Beneficial Effects

[0013] The present invention found that the serum vasostatin-2 level of patients with restenosis after coronary angioplasty was lower than that of patients without restenosis (P<0.001), and vasostatin-2 significantly improved neointimal hyperplasia and phenotypic conversion of vascular smooth muscle cells (VSMC) after injury. The mechanism is that vasostatin-2 binds to angiotensin-converting enzyme 2 (ACE2) and activates the downstream nuclear receptor subfamily 1 group D member 1 (NR1D1)-growth arrest specific 1 (Gas1) pathway, thereby promoting VSMC apoptosis and inhibiting its proliferation. ACE2 binding defective vasostatin-2 mutants and NR1D1 deficiency in VSMC weakened the beneficial effects of vasostatin-2 on neointimal hyperplasia. In summary, the present invention found that vasostatin-2 alleviated neointimal growth caused by vascular injury in mice and inhibited the proliferation of VSMCs in vitro through the ACE2 / NR1D1 / Gas1 pathway, making vasostatin-2 an effective strategy for treating vascular intimal hyperplasia diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 AB showed that serum vasostatin-2 levels were associated with vascular restenosis.

[0015] Figure 2 AL showed that vasostatin-2 attenuated injury-induced vascular intimal hyperplasia in mice.

[0016] Figure 3 AK showed that vasostatin-2 inhibited PDGF-BB-induced HASMC proliferation, migration and phenotypic switching.

[0017] Figure 4 AH showed that vasostatin-2 upregulated NR1D1 expression in vivo and in vitro.

[0018] Figure 5 AQ showed that NR1D1 deficiency partially reversed the inhibitory effect of vasostatin-2 on injury-induced vascular intimal hyperplasia.

[0019] Figure 6 AO showed that Gas1 is a direct downstream target of NR1D1 upon vasostatin-2 stimulation.

[0020] Figure 7 AR showed that vasostatin-2 exerts its effects by binding to ACE2 in vascular smooth muscle cells.

[0021] Figure 8 AW shows vasostatin-2 R72F / Q76F / K77FReversed the inhibitory effect of vasostatin-2 on neointimal hyperplasia. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0023] Example 1

[0024] 1. Experimental Methods

[0025] 1. Serum vasostatin-2 level detection:

[0026] Methods: A total of 5069 consecutive patients with CAD underwent percutaneous coronary intervention (PCI). Patients with a history of coronary artery bypass grafting (n = 365), acute coronary syndrome (n = 502), malignancy or immune system disease (n = 69), renal insufficiency requiring hemodialysis (n = 58), NYHA class III or IV heart failure (n = 109), and those lost to follow-up at 1 year (n = 252) were excluded to ensure consistency of serum data. 442 patients with restenosis and 442 patients without restenosis were identified by propensity score matching (including sex, age, body mass index (BMI), smoking, hypertension, diabetes, and dyslipidemia) and included in the final analysis. Blood samples were collected from all patients after an overnight fast on the day of angiography. Blood samples were collected at 8:00 am, immediately centrifuged to obtain serum, and stored at -80°C to mitigate the diurnal variation of vasostatin-2 concentrations and the significant effect of the fasting interval. Serum vasostatin-2 levels were measured by enzyme-linked immunosorbent assay (ELISA).

[0027] 2. Cell culture:

[0028] Primary mouse smooth muscle cells (mVSMC) were obtained from 8-10 week old mice as follows. After euthanasia of mice, the aorta was isolated and the connective tissue, endothelium and adventitia were removed. The intima was cut into 3-4 mm long fragments and then treated with 0.25% collagenase type II and 0.5% elastase type II at 37 ° C for 1 hour. DMEM / F12 medium supplemented with 1% penicillin / streptomycin solution and 10% FBS fetal bovine serum were used to promote mVSMC adhesion and proliferation. Primary human aortic smooth muscle cells (HASMC) were cultured in smooth muscle cell culture medium. All cell experiments used 4-6 generations of mVSMC and HASMC. HASMC was stimulated with platelet-derived growth factor (PDGF)-BB to promote its proliferation, migration and secretory phenotype. Cell proliferation was detected using CCK-8 assay, and smooth muscle cell migration was detected using transwell assay and scratch assay. Cell immunofluorescence and western blot assays were used to detect markers of vascular smooth muscle cell contraction and secretory phenotypes.

[0029] 3. Establishment of mouse femoral artery vascular injury model:

[0030] A femoral artery guidewire injury model was established using 8-12 week old male mice. First, mice were anesthetized with 2% isoflurane and placed on a heating pad to maintain their body temperature. The right femoral artery was exposed through a skin incision, and a guidewire was inserted through an arteriotomy. Next, the guidewire was repeatedly advanced and withdrawn distally several times to induce arterial injury. For perivascular treatment, pluronic gel (50 μL, 25% (w / v) P-127 Pluronic gel) containing recombinant human vasostatin-2 (4 μM, 25% pluronic gel) or saline was applied to the adventitia of the vessel. The incision was sutured after hemostasis. After surgery, the recovery of the mice was monitored. After the specified time, the mice were euthanized and the femoral arteries were collected for histological analysis. In vitro release experiments were performed using pluronic gels containing different concentrations of vasostatin-2, and in vivo experiments were performed using 25% pluronic gels supplemented with 4 μM vasostatin-2, because the release concentration of vasostatin-2 under this condition was equivalent to the stimulating concentration in cell experiments.

[0031] 4. Generation of conditional knockout mice:

[0032] Male mice aged 8-12 weeks were used in this study. VSMC-specific nuclear receptor subfamily 1 group D member 1 (Nr1d1) knockout mice (Nr1d1 flox / flox Tagln-Cre, Nr1d1 ΔSMC ) is obtained by converting Nr1d1 floxedMice were crossed with Tagln-Cre mice from the Jackson Laboratory. flox / flox As a control.

[0033] 5. Immunofluorescence staining:

[0034] For immunofluorescence staining, the sections were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 10% goat serum, and incubated with the corresponding primary antibodies overnight at 4°C. Subsequently, fluorescently labeled secondary antibodies were added in the dark at 37°C, and the sections were counterstained with DAPI. Finally, the stained sections were visualized and imaged under a fluorescence microscope.

[0035] 6. Western blot analysis:

[0036] After pretreatment, HASMC was lysed with lysis buffer. The lysed samples were separated by electrophoresis with equal loading. After electrophoresis, the protein samples in the gel were transferred to a polyvinylidene difluoride membrane. Then, the membrane was blocked in 5% skim milk and subsequently incubated with the primary antibody overnight at 4°C. After washing three times with PBS and incubating with the HRP-labeled secondary antibody for 1h, the protein bands were detected using an electrochemiluminescence system.

[0037] 7. Immunoprecipitation (IP) and co-immunoprecipitation (co-IP):

[0038] HASMCs were stimulated with His-tagged vasoststin-2, while HEK-293 cells overexpressing Flag-ACE2 or its truncated mutants were stimulated with His-tagged vasostatin-2. Lysates were collected and homogenized using IP lysis buffer and a phosphatase inhibitor cocktail. Next, the protein concentration of each sample was determined by centrifugation at 13,000 × g for 20 minutes. Subsequently, 200 μg of protein lysate was incubated with 2 μg of anti-ACE2 or anti-His antibody at 4 ° C for 12 hours with continuous rotation. The bead-antibody-antigen complex was separated using a magnetic separator and washed twice with IP lysis buffer. The antigen was subsequently eluted and denatured by boiling with 30 μL 1× SDS loading buffer at 100 ° C for 10 minutes, followed by SDS-PAGE analysis.

[0039] 8. ACE2 activity fluorescence assay and angiotensin 1-7 (Ang1-7) detection:

[0040] HASMC were starved overnight in 1% FBS and then treated with saline or vasostatin-2 for 24 hours under angiotensin II (1 μM) stimulation. The cells were then assayed for ACE2 activity. The substrate was added to the cell lysate and mixed immediately. Fluorescence was measured using a fluorescent microplate reader. Cell supernatants under different stimulations were collected and Ang1-7 levels were assessed by ELISA.

[0041] 9. Molecular docking:

[0042] Homology modeling was performed using SwissModel, and protein docking of ACE2 was downloaded from the PDB database (Protein Data Bank [PDB] ID: 1R42). The protein structure was cleaned by removing water molecules, ions, and other non-protein entities. A three-dimensional mesh around the ACE2 protein was created as a reference for docking calculations. The docking process between ACE2 and vasostatin-2 was simulated using HDOCK. The resulting docking poses were evaluated using a scoring function to rank the potential binding modes according to their binding affinity and interaction energy. Finally, the docked complex was analyzed to understand the key residues involved in the ACE2-vasostatin-2 interaction and the structural basis of its binding specificity.

[0043] 2. Experimental Results

[0044] 1. Serum vasostatin-2 levels are significantly correlated with vascular restenosis

[0045] The final analysis included 442 patients with restenosis and 442 patients without restenosis. Patients with restenosis had higher levels of fasting blood glucose, hsCRP, and the proportion of vascular lesions (such as B2 / C lesions, bifurcation lesions, chronic total occlusions, and multivessel lesions) than those without restenosis. In addition, the eGFR, HDL cholesterol levels, and ejection fraction of patients with restenosis were significantly lower than those of patients without restenosis. It is worth noting that the serum vasostatin-2 levels of patients with restenosis were significantly lower than those of patients without restenosis (2.23 (0.94-4.36) vs. 4.14 (2.52-5.39) ng / mL, P<0.001)( Figure 1 A). According to the restricted cube plot results, serum vasostatin-2 levels were nonlinearly associated with the risk of restenosis (P<0.001). When serum vasostatin-2 levels were below 3.32 ng / mL, they were significantly negatively correlated with the occurrence of restenosis after adjusting for age, sex, smoking status, BMI, eGFR, HbA1c, hsCRP, and hypertension. When the serum vasostatin-2 level exceeded 3.32 ng / mL, further increasing the serum vasostatin-2 concentration did not show significant additional benefits ( Figure 1 B).

[0046] 2. Vasostatin-2 reduces injury-induced angiogenesis intimal hyperplasia in mice

[0047] To investigate the effect of vasostatin-2 on injury-induced neointimal hyperplasia, a mouse femoral artery guidewire injury model was established. Immediately after injury, mice were injected with PF-127 gel containing vasostatin-2 or saline ( Figure 2 A). Histological staining of vascular tissue 28 days after injury showed that vasostatin-2 administration significantly reduced neointimal hyperplasia and intima-media ratio compared with the control group (all P<0.05) ( Figure 2 B-2D). Masson's trichrome staining showed that collagen deposition was reduced in mice treated with vasostatin-2 ( Figure 2 In addition, mice treated with vasostatin-2 showed less inflammation than control mice, as evidenced by fewer CD11B+ and F4 / 80+ positive cells ( Figure 2 G-2I). α-SMA and Ki67 immunofluorescence staining further demonstrated that vasostatin-2 administration reduced smooth muscle cell proliferation in damaged arteries ( Figure 2 J-2L). Taken together, these results demonstrate that vasostatin-2 attenuates neointimal hyperplasia following vascular injury.

[0048] 3. Vasostatin-2 inhibits HASMC proliferation and migration and suppresses the secretory phenotype in HASMC

[0049] To evaluate the effect of vasostatin-2 on VSMC proliferation and migration, HASMC were stimulated with increasing concentrations of vasostatin-2 under PDGF-BB exposure conditions, as shown in Figure 3 As shown in A, vasostatin-2 inhibited HASMC proliferation in a concentration-dependent manner. Scratch assay and Transwell assay showed that vasostatin-2 inhibited HASMC migration in a concentration-dependent manner ( Figure 3 B-3E). The vasostatin-2 concentration in subsequent in vitro experiments was 40ug / mL. Vasostatin-2 stimulation reduced the level of fibrosis in HASMC (represented by the reduction of MMP2, MMP9, Col1a1 and Col3a1 levels) ( Figure 3 F), and inhibited the transition of VSMCs from a contractile phenotype to a secretory phenotype (represented by increased levels of contractile phenotype markers α-SMA, SM-MHC, and SM22-α and decreased levels of secretory phenotype marker OPN) ( Figure 3G). Immunofluorescence staining of HASMC showed that Ki67+HASMC was significantly reduced after vasostatin-2 stimulation, further indicating that it has an inhibitory effect on HASMC proliferation ( Figure 3 H and 3I). In addition, double immunofluorescence staining of contractile (α-SMA) and secretory (OPN) phenotype markers showed that α-SMA expression was enhanced and OPN expression was decreased in vasostatin-2-treated HASMCs compared with the saline group ( Figure 3 J and 3K). These results indicate that vasostatin-2 inhibits PDGF-BB-induced HASMC proliferation and migration and suppresses the phenotypic transition of HASMC.

[0050] 4. Vasostatin-2 upregulates NR1D1 expression in injury-induced vascular tissue and HASMC

[0051] To elucidate the protective mechanism of vasostatin-2 in inhibiting neointimal hyperplasia, RNA sequencing analysis was performed on the femoral arteries of mice (administered vasostatin-2 or saline) 3 days after wire injury ( Figure 4 A). Gene ontology enrichment analysis showed that vasostatin-2 affected several key processes of vascular remodeling, including wound healing, cell chemotaxis, and extracellular matrix organization ( Figure 4 B). Among the differentially expressed genes (DEGs), the significant upregulation of NR1D1 was noteworthy ( Figure 4 C). This gene showed the most significant upregulation among all members of the nuclear receptor superfamily affected by vasostatin-2 ( Figure 4 D). Western blotting and RT-qPCR showed that vasostatin-2 significantly increased the expression of NR1D1 in the presence or absence of PDGF-BB stimulation ( Figure 4 E and 4F). Immunofluorescence staining of HASMC confirmed that NR1D1 expression increased under vasostatin-2 stimulation ( Figure 4 G). In addition, immunofluorescence double staining of α-SMA and NR1D1 showed that NR1D1 expression in VSMCs was significantly increased after vasostatin-2 treatment in the injured femoral artery ( Figure 4 H).

[0052] 5. VSMC-specific NR1D1 deficiency partially reverses the inhibitory effect of vasostatin-2 on injury-induced vascular intimal hyperplasia

[0053] To investigate whether NR1D1 mediates the inhibitory effect of vasostatin-2 on intimal hyperplasia, we used NR1D1 knockout mice (Nr1d1 ΔSMC ) and littermate control mice (Nr1d1 flox / flox ) were given vasostatin-2, and histological examination was performed 28 days after injury. Hematoxylin and eosin (HE) staining results showed that Nr1d1 ΔSMC Mice given vasostatin-2 showed more pronounced neointimal hyperplasia and a larger intima-media ratio ( Figure 5 A-5C). In immunofluorescence, Nr1d1 ΔSMC Mice showed increased α-SMA-positive areas and Ki67+ cells ( Figure 5 F-5I). Masson staining showed Nr1d1 ΔSMC Collagen deposition in mice is higher than that in Nr1d1 flox / flox Mice were more obvious ( Figure 5 D and 5E). These histological findings suggest that NR1D1 mediates the effect of vasostatin-2 in inhibiting injury-induced vascular neointimal hyperplasia in vivo.

[0054] Consistently, in vitro experiments showed that silencing NR1D1 significantly attenuated vasostatin-2-induced inhibition of HASMC proliferation and migration ( Figure 5 J-5L). Immunofluorescence showed that under the stimulation of vasostatin-2, the fluorescence intensity of OPN in HASMCs of the si-NR1D1 group was stronger than that in the control cells, and the fluorescence intensity of α-SMA was weaker than that in the control cells ( Figure 5 N and 5O). Western blot results also showed that the expression of contractile phenotype markers (α-SMA, SM-MHC, SM22α) was reduced and the expression of secretory phenotype markers (OPN) was increased in HASMCs in the si-NR1D1 group. In addition, fibrosis-related markers (MMP2, MMP9, Col1a1, and Col3a1) were significantly upregulated in these cells ( Figure 5 P and 5Q).

[0055] 6. Identification of Gas1 as a direct target of NR1D1 by integrating CUT&Tag and RNA sequencing analysis

[0056] To determine the relationship between NR1D1-regulated genes and the downstream pathway effects of vasostatin-2, CUT&Tag assays were performed on primary mouse vascular smooth muscle cells (mVSMCs) treated with saline or vasostatin-2. The peak calling software MACS2 was used for the analysis, and 8816 and 7377 peaks were identified in vasostatin-2-treated and saline-treated samples, respectively ( Figure 6 A). Peak annotation analysis was performed using bedtools to identify regions in vasostatin-2 that were associated with NR1D1 in VSMCs. The results showed that 49.33% and 33.09% of the peaks in the saline and vasostatin-2 groups, respectively, mapped to the promoter transcription start site (TSS) ±3kb combined region, indicating that they may be involved in NR1D1 transcriptional regulation ( Figure 6 B). HOMER analysis revealed the top two binding motifs of NR1D1 upon vasostatin-2 stimulation ( Figure 6 C). ClusterProfiler was further used to perform pathway enrichment analysis on differentially annotated genes based on the KEGG database. Notably, vasostatin-2 significantly upregulated the vascular smooth muscle cell contraction pathway through NR1D1 regulation, which was consistent with the observed cell phenotype ( Figure 6 D [upper]). Under vasostatin-2 stimulation, NR1D1-regulated genes were significantly enriched in pathways such as apoptosis, mitochondrial autophagy, and focal adhesion ( Figure 6 D [lower]). To identify the direct downstream targets of NR1D1, the CUT&Tag and RNA sequencing results were comprehensively analyzed, and five candidate target genes including Gas1 were obtained by intersecting all peak annotated genes under vasostatin-2 stimulation with the DEGs in RNA sequencing ( Figure 6 E). The differential binding ability of Gas1 to NR1D1 under saline and vasostatin-2 stimulation was tested using genomic locus analysis ( Figure 6 F). Subsequently, the correlation between Gas1 and NR1D1 expression was verified. In HASMC, vasostatin-2 treatment upregulated Gas1, while knockdown of NR1D1 downregulated its expression ( Figure 6 G and 6H). In immunofluorescence staining, Nr1d1 ΔSMC Gas1 is also downregulated in mice ( Figure 6I). These results indicate that Gas1 expression increases with increased NR1D1 expression. Luciferase reporter gene assays in HEK-293 cells and CUT&RUN-qPCR in HASMCs showed that NR1D1 overexpression increased Gas1 promoter activity, while vasostatin-2 enhanced the transcriptional activation of Gas1 by NR1D1 ( Figure 6 J and 6K). Given the role of Gas1 in promoting apoptosis and inhibiting the cell cycle, and the fact that NR1D1-regulated genes were significantly enriched in apoptotic pathways upon vasostatin-2 stimulation, apoptosis in HASMCs was assessed using flow cytometry and TUNEL staining was performed on paraffin sections of injured femoral arteries. Both results indicated that vasostatin-2 promoted VSMC apoptosis, and NR1D1 deficiency partially inhibited the pro-apoptotic effect of vasostatin-2 ( Figure 6 L and 6M). Cell cycle analysis of HASMC also showed that after vasostatin-2 stimulation, G0 / G1 increased and S phase cells decreased, and NR1D1 knockdown could partially reverse this phenomenon ( Figure 6 N and 6O). These results suggest that NR1D1 transcriptionally activates Gas1, thereby promoting apoptosis and inhibiting VSMC proliferation.

[0057] 7. Vasostatin-2 binds to ACE2 and acts through the ACE2 / MasR / PPARγ / NR1D1 / Gas1 axis

[0058] To identify the direct binding targets of vasostatin-2 in VSMCs, GST pull-down and LC-MS / MS analysis were performed using recombinant GST-tagged vasostatin-2 (GST-VS2) and GST vector (GST-con) as a control ( Figure 7 A). Three potential proteins that specifically bind to GST-VS2 were identified in human VSMCs, and only ACE2 was localized to the cell membrane ( Figure 7 B and 7C). In addition, vasostatin-2 has a specific docking pose with ACE2 (PDB: 1R42) and a binding energy of -228.45 kcal / mol. The bonds formed between vasostatin-2 and ACE2 include His54–Ser280, Leu58–Asn154, Arg72–Tyr127, Gln76–Ser124, and Lys77–Tyr50, which play a crucial role in the interaction between the two proteins ( Figure 7D). Immunofluorescence staining showed that vasostatin-2 and ACE2 co-localized on the HASMC cell membrane. Co-immunoprecipitation and truncation mutation experiments further verified that vasostatin-2 and ACE2 bound to each other ( Figure 7 E and 7F), the binding domain is located at the N-terminus of ACE2 ( Figure 7 G). It is well known that ACE2 degrades angiotensin II to angiotensin 1-7. Angiotensin 1-7 triggers Mas receptor (MasR)-mediated signaling pathways, exerting vasodilatory and anti-inflammatory effects. Therefore, the enzymatic activity of ACE2 and angiotensin 1-7 levels in VSMCs under vasostatin-2 exposure were evaluated to determine whether vasostatin-2 affects ACE2 activity and downstream gene expression. Compared with saline treatment, vasostatin-2 treatment increased ACE2 activity and angiotensin 1-7 levels in HASMCs after angiotensin II stimulation ( Figure 7 H and 7I). Predictions from the UCSC website indicate that PPARγ transcriptionally regulates NR1D1. Previous RNA sequencing data also showed that PPARγ expression was upregulated after vasostatin-2 treatment, indicating that PPARγ may be upstream of NR1D1 in the action of vasostatin-2. Therefore, it is hypothesized that vasostatin-2 exerts an inhibitory effect on VSMC proliferation, migration, and phenotypic transition through the ACE2 / MasR / PPARγ / NR1D1 / Gas1 axis. Using siRNA to knock down ACE2, PPARγ, NR1D1, and Gas1, respectively, Western blotting showed that there was a clear upstream-downstream relationship within the ACE2 / PPARγ / NR1D1 / Gas1 pathway ( Figure 7 J-7Q). In conclusion, the above experiments indicate that vasostatin-2 inhibits neointimal hyperplasia through the ACE2 / MasR / PPARγ / NR1D1 / Gas1 axis ( Figure 7 R).

[0059] 8. Mutant vasostatin-2 R72F / Q76F / K77F Reduced protective effect against neointimal hyperplasia in mice

[0060] In order to identify the key binding domain of vasostatin-2 in inhibiting intimal hyperplasia, the following two vasostatin-2 mutants were constructed based on the molecular docking results: VS2mut1 (Vasostatin-2 H54F / L58F ) and VS2mut2 (Vasostatin-2 R72F / Q76F / K77F). The results of HASMCs immunoprecipitation showed that VS2wt and VS2mut1 had similar binding abilities to ACE2, and the binding ability of VS2mut2 to ACE2 was significantly lower than that of VS2wt and VS2mut1 ( Figure 8 A). In the ACE2 activity assay and angiotensin 1-7 detection assay, the effect of VS2mut2 in the cell supernatant was significantly lower than that of VS2wt ( Figure 8 B and 8C), the effect of VS2mut2 in the cell supernatant was significantly lower than that of VS2wt ( Figure 8 B and 8C). Therefore, VS2mut2 can be used as a negative control to verify the effects and pathways of angiostatin-2. Compared with VS2wt, VS2mut2 did not change the expression level of ACE2, but significantly inhibited the expression pathway of PPARγ / NR1D1 / Gas1 ( Figure 8 D), indicating that Lys-77, Gln-76, and Arg-72 of angiostatin-2 are key residues for ACE2 binding and triggering the ACE2 / MasR / PPARγ / NR1D1 / Gas1 pathway. Subsequently, cell proliferation and migration assays of VS2wt and VS2mut2 showed that the effect of VS2mut2 was significantly weakened compared with VS2wt ( Figure 8 E-8I). In addition, VS2mut2 increased the levels of secretory phenotype proteins (MMP-2 and MMP-9) and OPN, while inhibiting the expression of contractile phenotype proteins (α-SMA, SM-MHC, and SM22-α), as determined by Western blot examination ( Figure 8 J and 8K). Immunofluorescence further confirmed that VS2mut2 had a lower inhibitory effect on cell proliferation than VS2wt, and the shift to a secretory phenotype was more significant ( Figure 8 L-8O). In vivo, VS2wt and VS2mut2 were topically applied to the injured femoral artery and their effects were observed 28 days after injury. Notably, VS2mut2 increased neointimal hyperplasia and intima-media ratio ( Figure 8 P and 8Q). In addition, Masson staining showed that collagen deposition was more obvious in injured arteries of VS2mut2-treated mice ( Figure 8 R and 8S). Immunofluorescence staining of α-SMA and Ki67 consistently showed that VS2mut2 promoted VSMC proliferation after injury compared with VS2wt ( Figure 8 T-8W). Collectively, these results suggest that VS2mut2 is less potent than VS2wt in suppressing intimal hyperplasia after vascular injury, emphasizing the importance of angiostatin 2 binding to ACE2 for its effects.

Claims

1. The use of a bioactive peptide in the preparation of a drug for treating intimal hyperplasia after vascular injury, characterized in that: The bioactive peptide is vasostatin-2.

2. The use according to claim 1, characterized in that: The vasostatin-2 reduces neointimal growth caused by vascular injury in mice and inhibits the proliferation of VSMCs in vitro through the ACE2 / NR1D1 / Gas1 pathway.

3. The use according to claim 1, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

4. The use according to claim 1, characterized in that: The pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, and a disintegrant.

5. A pharmaceutical composition for treating intimal hyperplasia after vascular injury, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of vasostatin-2 according to claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that: The dosage form of the pharmaceutical composition includes at least one of tablets, pills, powders, solutions, suspensions, emulsions, and granules.

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