M1 Macrophage-Derived Exosomal miR-155-5p Exacerbates Aortic Dissection Progression via SMAD5-Mediated Regulation of Vascular Smooth Muscle Cell Phenotype

M1 Macrophage-Derived Exosomal miR-155-5p Exacerbates Aortic Dissection Progression via SMAD5-Mediated Regulation of Vascular Smooth Muscle Cell Phenotype | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article M1 Macrophage-Derived Exosomal miR-155-5p Exacerbates Aortic Dissection Progression via SMAD5-Mediated Regulation of Vascular Smooth Muscle Cell Phenotype Dengwei Cao, Xinyi Li, Shaoping Zhu, Jianfeng Chen, Haoxiang Li, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8986811/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Aortic dissection (AD) constitutes a critical and potentially lethal cardiovascular emergency, yet its underlying pathogenesis remains largely obscure. The present research aimed to elucidate the contribution of exosomes derived from M1 macrophages—particularly those carrying a high load of miR-155-5p—to the exacerbation of AD. Methods Macrophage infiltration and exosome distribution were evaluated in human and mouse AD tissues. Exosomes were isolated from M1-polarized RAW264.7 macrophages and characterized. Primary mouse VSMCs were treated in vitro, while a β-aminopropionitrile (BAPN)--established murine AD model was used for in vivo studies. Interventions included GW4869 and engineered exosomes loaded with Antago-miR-155-5p. Techniques included qRT-PCR, Western blotting, luciferase assays, RNA-FISH, and histological analyses. Results M1 macrophages and their exosomes were markedly enriched in AD tissues and colocalized with macrophage markers. M1-derived exosomes were internalized by VSMCs, significantly downregulated contractile indicators (CNN, α-SMA, MYOCD, SM22α), and induced synthetic phenotypic switching via exosomal miR-155-5p targeting SMAD5 (luciferase assay). In BAPN-induced mice, inhibition of exosome secretion (GW4869) or treatment with Antago-miR-155-5p-loaded exosomes significantly reduced AD incidence (from 86.7% to 46.7%), mortality, aortic dilation, elastic fiber fragmentation, and restored contractile marker and SMAD5 expression. Conclusions Exosomal miR-155-5p originating from M1 macrophages facilitates AD development through targeting SMAD5 and driving VSMC phenotypic switching. Engineered Antago-miR-155-5p exosomes represent a novel, effective therapeutic strategy for mitigating AD, providing a foundation for exosome-based RNA interference in vascular diseases. Aortic dissection Exosomes Vascular smooth muscle cell miR-155-5p SMAD5 Phenotypic transition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Aortic dissection (AD) represents a lethal vascular condition involving the architectural degradation of the aortic wall.[ 1 , 2 ]. While operative management persists as the cornerstone of clinical therapy, postoperative mortality is still high. Currently, effective non-surgical strategies for reducing mortality associated with AD are insufficient [ 3 – 7 ]. Therefore, elucidating the mechanisms underlying AD progression, discovering new therapeutic targets, and identifying biomarkers for early diagnosis are crucial for improving early intervention and personalized treatment approaches. Among the various pathological processes involved in AD, inflammation and vascular remodeling are considered central. Accumulating evidence highlights critical roles for inflammatory cell infiltration [ 8 , 9 ], extracellular matrix (ECM) degradation [ 10 ], and VSMC phenotypic transition in AD initiation and progression [ 11 , 12 ]. Among the infiltrating inflammatory cells, macrophages are pivotal. Under pathological stimuli such as hypertension, they become activated and often polarize toward the pro-inflammatory M1 phenotype [ 13 , 14 ]. These macrophages further aggravate aortic injury and vascular remodeling through the secretion of inflammatory mediators and matrix metalloproteinases (MMPs), which compromise the structural integrity of the extracellular matrix (ECM) and accelerate AD progression [ 15 , 16 ]. Moreover, inflammatory factors or mechanical stress can prompt VSMCs to switch from contractile to synthetic phenotypes [ 17 – 19 ]. This phenotypic transition reduces their contractile function while increasing proliferation, migration, and synthetic activities, collectively leading to deterioration of the aortic wall [ 20 ]. Notably, intercellular communication between macrophages and VSMCs is vital in AD pathogenesis [ 21 ]. Although previous studies suggested that M1 macrophages affect VSMC phenotype switching and vascular integrity [ 22 ], the precise molecular mechanisms of this interaction remain unclear. Emerging evidence indicates that exosomes function as essential vehicles for orchestrating cross-talk among cells during cardiovascular disorders by transferring bioactive cargos, including miRNAs, over long distances [ 23 – 25 ]. While macrophage-derived exosomes are known to participate in the pathogenesis of vessel-related disorders, including atherosclerosis and neointimal hyperplasia via miRNAs (e.g., miR-21-3p, miR-222) [ 26 , 27 ], their contribution to AD progression—and in particular the specific miRNA effectors regulating VSMC phenotype in vivo—remains largely unexplored. Notably, Notably, how M1 macrophage-sourced exosomal miRNAs directly regulate the SMAD signaling pathway in VSMCs remains an open question. In this work, we provide the first evidence that that M1 macrophage-derived exosomes deliver miR-155-5p to VSMCs, where it targets and suppresses SMAD5, thereby promoting synthetic phenotypic switching and exacerbating AD progression. Furthermore, we developed an engineered exosome-based delivery system carrying Antago-miR-155-5p, which effectively mitigates disease severity in a BAPN-induced mouse model. These findings uncover a novel macrophage-VSMC communication axis in AD pathogenesis and provide a promising foundation for exosome-mediated RNA interference as a precise clinical intervention. 2. Materials and methods 2.1 Materials Cell culture and transfection reagents: The essential media and supplements for cell maintenance, including high-glucose Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin-EDTA, and 100× penicillin-streptomycin, were procured from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). For transfection purposes, Lipofectamine 3000 and Opti-MEM™ I Reduced-Serum Medium were acquired through Invitrogen and Gibco (Thermo Fisher Scientific). The miR-155-5p mimics (including FAM-tagged versions), Antago-miR-155-5p, and respective negative control sequences (NC mimics and Antago-NC) were custom-synthesized by RiboBio Co., Ltd. (Guangzhou, China). The SMAD5-expressing plasmid was provided by Tsingke Biotechnology Co., Ltd. Macrophage polarization and stimulation reagents: Lipopolysaccharide (LPS, catalog no. BS904) was purchased from Biosharp (Shanghai, China). Recombinant mouse interferon-gamma (IFN-γ, catalog no. HY-P70610) was from MedChemExpress (Monmouth Junction, NJ, USA). Angiotensin II (Ang II; cat. no. A9525) was sourced through Sigma-Aldrich (Shanghai, China).Regarding exosome-related materials, the secretion inhibitor GW4869 was acquired from MedChemExpress, while the PKH26 Red Fluorescent Cell Linker Kit was provided by Yu Jiubo Co., Ltd. (Shanghai, China). Antibodies: Specific primary antibodies targeting CD9, CD63, TSG101, Calponin (CNN), and inducible nitric oxide synthase (iNOS) were purchased via Abcam (Cambridge, UK). Antibodies against SM22α, CD68, F4/80, α-SMA, and CD206 were from Proteintech Group, Inc. (Wuhan, China). The anti-MYOCD antibody was sourced from Sigma-Aldrich; furthermore, ABclonal Technology Co., Ltd. (Wuhan, China) provided the antibodies for SMAD5, E2F2, and SATB1. Other reagents:Protease and phosphatase inhibitor cocktails were from Sigma-Aldrich. TRIzol reagent was from Invitrogen.β-aminopropionitrile monofumarate (BAPN, catalog no. S42995) was from Yuanye Bio-Technology (Shanghai, China). 2.2 Human tissue study Specimens of aortic tissue, comprising both sporadic aortic dissection cases and healthy controls, were retrieved from our institutional biobank. We excluded individuals presenting with hereditary aortic conditions (e.g., Marfan syndrome) or dissections involving the mitral or aortic valves to maintain cohort homogeneity. For the control group, aortic tissues were harvested during cardiac transplantations from age-matched donors exhibiting no history of aortic coarctation, aneurysm, dissection, or prior surgical intervention. This study adhered to the Declaration of Helsinki ethical standards, with all protocols involving human material receiving formal endorsement from the Human Research Ethics Committee at Zhongnan Hospital, Wuhan University(Approval No. 2023187). 2.3 Animal study All animal-related experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals. The study design underwent ethical review and approval by the Animal Care and Use Committee of Zhongnan Hospital, Wuhan University (No. ZN2023009). The animals were maintained under a standard light/dark cycle, with ad libitum access to standardized chow and water. To establish the AD model, three-week-old male mice received 1 g/kg/day of BAPN [ 15 , 28 ] (Yuanye Bio-Technology, Shanghai, China) via their drinking supply over a 4-week duration. Mice were allocated into experimental cohorts (n = 15 per group) through a randomization process. Treatment delivery was performed through either intraperitoneal (i.p.) injection or intravenous (i.v.) infusion every 48 hours from week 3 to week 7 (end of experiment).GW4869 intervention Groups: Saline + DMSO, Saline + GW4869, BAPN + DMSO, BAPN + GW4869. GW4869 (20 µM stock, diluted appropriately) or vehicle (DMSO) was given i.p.Exosome administration Groups: Saline + M0-Exos, Saline + M1-Exos, BAPN + M0-Exos, BAPN + M1-Exos.Purified M0-Exos or M1-Exos (20 µg protein in 150 µL PBS) were injected via tail vein.Antago-miR-155-5p exosome therapy Groups: BAPN + Antago-miR-NC-Exos, BAPN + Antago-miR-155-5p-Exos.Engineered exosomes (M0-derived, loaded with Antago-miR-155-5p or negative control, 20µg protein/150 µL) were administered i.v. via tail vein. 2.4 Isolation and culture of primary mouse VSMCs Under sterile conditions, thoracic aortic tissues were harvested from male C57BL/6 mice at 10 weeks of age[ 29 ]. After euthanasia (cervical dislocation) and 70% ethanol immersion, the aorta was perfused with PBS via left ventricular puncture, dissected, and adventitia/fat removed under a stereomicroscope. Cleaned aortas were cut into 1–2 mm² pieces in serum-free high-glucose DMEM, following a 60-minute enzymatic digestion (0.25% trypsin and collagenase) maintained at 37°C, the reaction was neutralized using 10% FBS-supplemented DMEM. After centrifugation, the resulting cell pellet was homogenized in complete growth medium and subsequently distributed into six-well plates pre-coated with 0.1% gelatin.The primary cultures were maintained in a humidified 5% CO2 environment at 37°C. Initial media replacement occurred after a 3-day quiescent period, followed by regular changes every 72 hours. Experimental assays utilized cells between the second and third passages. 2.5 Cell culture and transfection The RAW264.7 cells were procured from the Cell Bank in Shanghai, China, which is part of the Chinese Academy of Sciences. Mice at eight weeks of age had their thoracic aortas used to generate VSMCs. We cultured RAW264.7 cells and primary VSMCs in high-glucose DMEM with 10% FBS and 1% P/S. Additional analysis was conducted using VSMCs collected at passages two to three. Cells were serum-starved overnight in DMEM with 0.1% FBS, then treated with 1 µM Angiotensin II (Ang II, Sigma, #A9525) for 24 hours prior to experiments. To distribute miR-155-5p mimics (50 nM), miR-155-5p inhibitors (100 nM), or SMAD5 plasmids (2 µg/mL), transfection was carried out using Lipofectamine 3000 (Invitrogen, #L3000015) at a cell density of about 60%. 2.6 Induction of M0 and M1 macrophages RAW264.7 macrophages were maintained in high-glucose DMEM containing 1% penicillin-streptomycin and 10% FBS. Upon achieving a confluency of nearly 60%, the growth medium was replaced by DMEM supplemented with 10% exosome-depleted FBS. To induce M1 polarization, macrophages were stimulated for a 24-hour period using 20 ng/mL IFN-γ (MedChemExpress, #HY-P70610) and 100 ng/mL LPS (Biosharp, #BS904). Collecting macrophages for phenotypic evaluation and observing cell morphology following activation. 2.6. Exosome purification Following recognized protocols, differential ultracentrifugation was employed to isolate exosomes from the collected culture supernatants. RAW264.7 macrophages were plated uniformly on 150-mm culture plates. At approximately 60% confluency, the medium was exchanged for DMEM containing 10% exosome-free FBS. After a 24-hour activation with 20 ng/mL IFN-γ and 100 ng/mL LPS, the cells underwent a medium exchange to serum-free DMEM for an additional 48-hour incubation. The harvested conditioned media was initially subjected to centrifugation at 300 ×g (10 min, 4°C), followed by a subsequent round at 2,000×g (30 min, 4°C) to eliminate cellular debris and detached cells. After passing the supernatant through a 0.22 µm membrane filter (Merck Millipore), the filtrate was further purified via centrifugation at 10,000×g for 30 minutes at 4°C. Final exosome recovery was achieved by ultracentrifugation (Beckman Coulter SW28 rotor) at $ 120,000×g for 90 minutes at 4°C. The resulting pellet was then resuspended in 100 µL of sterile PBS. 2.7. Transmission electron microscopy (TEM) imaging To analyze the shape of the separated exosomes, TEM was used. The exosome suspension, which was 20 µL in volume, was spread out on copper grids and left to incubate for 5–10 minutes. The grids were sponged with filter paper to soak up any extra fluid, allowed to air dry for a short while, and then negatively stained with 20 µL of 2% phosphotungstic acid for 5 minutes. After removing any excess staining solution, the grids were carefully dried under an incandescent bulb. The images were taken using an 80 kV accelerating voltage on a Hitachi H-7650 TEM (Japan). 2.8. Nanoparticle tracking analysis (NTA) NTA was performed with a ZetaView PMX110 (Particle Metrix) to assess exosome size and concentration. Prior to analysis, the chamber underwent triple rinsing with purified water. In order to calibrate the measuring chamber, exosome samples were diluted in PBS and then added. After the calibration, the chamber was filled with 1–2 µL of a diluted exosome solution in 2 mL of PBS. A 405 nm laser was used to take measurements while a 60-second video was recorded at 30 frames per second. This allowed for the ability to follow particles in real-time. Particle concentration and size distribution were analyzed by ZetaView software (version 8.02.28), utilizing Brownian motion principles. 2.9. Western blot (WB) for exosomes Exosomes were characterized by WB using antibodies against exosomal markers CD63, CD9, and TSG101 (all Abcam, 1:1000). Standard procedures were followed for protein extraction, electrophoresis, transfer, and immunoblotting. 2.10. PKH26 labeling and exosome uptake by VSMCs Isolated exosomes were fluorescently labeled using PKH26 dye, after which they were incubated with VSMCs for 12 hours. Cellular uptake of labeled exosomes was visualized through confocal microscopy. 2.11. Non-contact co-culture of RAW264.7 and VSMCs A non-contact Transwell co-culture assay was established using 0.4 µm polyethylene membrane inserts (Corning). We seeded RAW264.7 macrophages into the apical compartments, whereas VSMCs occupied the basal chambers of the Transwell system. Upon achieving approximately 60% confluency. Activation of RAW264.7 cells was achieved by exposure to 20 ng/mL IFN-γ, 100 ng/mL LPS, and 20 µM GW4869 (MCE), whereas PBS treatment was applied as a control. Following a 24-hour interval, the existing medium was swapped for serum-depleted high-glucose DMEM, extending the incubation period by an additional 48 hours. Subsequently, VSMCs were harvested for further evaluation. 2.12. miRNA sequencing and differential expression analysis We isolated total RNA from both M0/M1-polarized macrophages and their respective exosomal fractions. Evaluation of RNA integrity and concentration was performed via an Agilent 2200 Bioanalyzer. Shanghai Huaying Biotechnology (Shanghai, China) handled library construction and subsequent miRNA profiling, utilizing the Illumina HiSeq 2500 system. Differential expression analysis of miRNAs was conducted using standard bioinformatics pipelines. Criteria for identifying significant miRNA dysregulation included an adjusted P-value below 0.05 and a minimum absolute log2-fold change of 1. To verify the levels of exosomal miR-155-5p, qRT-PCR was executed on a Bio-Rad CFX96 Real-Time PCR platform (Bio-Rad, USA) employing the Vazyme ChamQ SYBR Master Mix. 2.13. miR-155-5p translocation analysis To trace exosomal trafficking, RAW264.7 macrophages underwent an initial 48-hour transfection with FAM-labeled miR-155-5p mimics, subsequently followed by induction toward the M1 state. After recovering exosomes from the conditioned media, they were stained with PKH26 and added to VSMC cultures for a 12-hour incubation period. After fixation, cytoskeletal and nuclear staining were performed, and confocal microscopy was employed to visualize intracellular localization of FAM-miR-155-5p. In associated experiments, exosomes originating from M1 macrophages (M1-Exos) were permeabilized using 0.5% Triton X-100 for 5 minutes, succeeded by RNA degradation with RNase (20 U/µL) for one hour. After PBS rinses, the exosomes were subjected to ultracentrifugation at 120,000 × g for 90 minutes (4°C) to eliminate residual RNase and Triton X-100. Subsequently, miR-155-5p levels in recipient VSMCs were measured through qPCR. 2.14. Bioinformatics analysis RNA sequencing was performed on aortic tissues from BAPN-induced AD mice and age-matched controls by Shanghai Huaying Biotechnology Co., Ltd. (Shanghai, China). Identification of differentially expressed genes (DEGs) was performed through the limma and edgeR R toolkits. The selection criteria were defined as an adjusted P-value < 0.05 (applying Benjamini-Hochberg correction) and a |log2 fold change| of at least 1. miR-155-5p target genes were predicted using TargetScan (v7.2) and miRTarBase (v8.0), focusing on conserved 3’UTR binding sites. Candidate targets were obtained by intersecting the predicted targets with significantly downregulated DEGs from the RNA-seq dataset. To explore the biological roles of these overlapping gene candidates, we performed functional enrichment analysis via the Metascape platform, utilizing standard settings, including GO, KEGG, and Reactome databases. Statistical significance was assigned to enriched functional terms exhibiting an adjusted P-value below 0.05. 2.15. Luciferase reporter assay To construct the reporter vectors, either the wild-type (WT) 3’UTR of SMAD5, which harbors the forecast miR-155-5p binding site, or its mutant (Mut) counterpart with a scrambled seed sequence, was inserted into the pGL6-basic luciferase plasmid (Promega) at a position following the firefly luciferase gene. All constructs were verified by Sanger sequencing. Primary VSMCs underwent plating in 24-well clusters and were subsequently co-transfected upon reaching 60–70% density. The transfection mixture comprised 200 ng of WT/Mut reporter plasmids, a 50 nM concentration of miR-155-5p mimics (or NC mimics), and 20 ng of pRL-TK (Promega) serving as the endogenous reference, facilitated by Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours post-transfection, cell lysis was performed to facilitate the sequential determination of firefly and Renilla luciferase signal intensities through the Dual-Luciferase Reporter Assay System (Promega) via a GloMax luminometer (Promega). Normalization of firefly luciferase levels against Renilla luciferase was conducted... All assays were executed in triplicate and replicated a minimum of three independent times. 2.16. Immunofluorescence (IF) assay Tissue immunofluorescence: Aortic samples were initially preserved in 4% paraformaldehyde (PFA) for a 24-hour period, followed by embedding in paraffin and preparation of 5-µm thick slices. These sections then underwent deparaffinization using xylene, rehydration via descending ethanol concentrations, and heat-induced epitope retrieval within a pH 6.0 citrate buffer environment using microwave heating for 10–15 min. After blocking with 10% bovine serum albumin (BSA) diluted in PBS for 1 hour at ambient temperature. This was followed by an overnight incubation at 4°C with specific primary antibodies... After three PBST washing cycles, the specimens were exposed to corresponding fluorescent secondary antibodies for a duration of 2 hours at room temperature. For nuclear visualization, DAPI (1 µg/mL) was applied for 5 minutes. Slides were secured using an antifade mounting medium prior to visualization under either a Leica fluorescence or confocal microscope. We utilized ImageJ software to quantify the colocalization and fluorescence intensities. Cellular immunofluorescence: Primary VSMCs or treated cells grown on coverslips underwent fixation in 4% paraformaldehyde for a duration of 15 min at ambient temperature, followed by a 10-minute permeabilization step utilizing 0.2% Triton X-100 (diluted in PBS). Subsequent to a 1-hour blocking period in 10% BSA, the specimens were subjected to overnight incubation with primary antibodies at 4°C. Exposure to secondary fluorescent antibodies was performed for 2 h under room temperature conditions. A Leica confocal microscope was employed for image acquisition, with subsequent quantification conducted via ImageJ software. 2.17. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was isolated from the samples utilizing the TRIzol reagent (purchased from Invitrogen). A NanoDrop One spectrophotometer was utilized to evaluate both the quantity and optical purity of the extracted RNA, which is made by Thermo Fisher Scientific. The HiScript III RT SuperMix (Vazyme) and 1 µg reverse transcription was executed starting with a 1 µg template of total RNA. Quantitative PCR assays were carried out on a Bio-Rad CFX96 Real-Time PCR detection system, employing the ChamQ SYBR Master Mix (Vazyme). GAPDH served as the internal control for normalizing gene expression, which was subsequently determined via the $ 2^{-\Delta\Delta CT} $ analytical approach. 2.18. WB Protein extraction was achieved by lysis in RIPA buffer supplemented with a cocktail of phosphatase and protease inhibitors from cultured cells and aortic tissue samples. Protein concentrations were determined by employing the BCA protein assay kit. Proteins of the same amount were electrophoretically separated on SDS-PAGE gels before being deposited onto PVDF membranes. Following a 1-hour blocking step in 5% non-fat milk at ambient temperature, the blots were exposed to specific primary antibodies overnight while maintained at 4°C. Membranes were subsequently incubated with HRP-conjugated secondary antibodies for 1 hour following multiple rinse cycles in TBST buffer. The protein signals were detected through ECL chemiluminescence and the resulting band intensities were processed using ImageJ. 2.19. Statistical analysis Data processing and statistical assessments were executed utilizing GraphPad Prism (v10.0). Results are expressed as the mean ± standard deviation (SD), except where specifically noted. The Shapiro-Wilk test was employed to verify whether the data followed a normal distribution. Comparisons utilized independent Student’s t-tests, or one-way ANOVA coupled with Tukey’s multiple comparison tests. Correlation assessments were performed by calculating Pearson’s correlation coefficients. Differences were considered statistically significant when the p-value was below 0.05. Blinding and randomization strategies were systematically employed during experiments and result evaluations. *: P < 0.05, **: P < 0.01, ***: P < 0.001 and ****: P < 0.0001denote varying levels of statistical significance. 3. Results 3.1. Accumulation of M1-polarized Macrophages and Exosomes in Aortic Dissection (AD) Tissue Although exosomes are known mediators of intercellular communication in cardiovascular disease[ 30 ], their role in AD remains poorly understood. Therefore, IF staining was performed to analyze macrophage and exosome distribution in human and mouse AD tissues compared with normal controls. In human tissues, AD samples showed marked infiltration of macrophages (CD68+, red) and elevated expression of the exosomal marker TSG101 (green), with strong colocalization in merged images (Fig. 1 A). Quantification revealed significantly higher relative fluorescence intensity for both TSG101 (Fig. 1 B) and CD68 (Fig. 1 C) in AD tissues compared to controls. Similar findings were observed in mouse aortic tissues, where AD samples exhibited increased TSG101 (green) and macrophage marker F4/80 (red) expression, with evident colocalization (Fig. 1 D). Quantitative analysis confirmed significantly elevated fluorescence intensity for TSG101 (Fig. 1 E) and F4/80 (Fig. 1 F) in AD versus control tissues. Further, macrophage polarization was further assessed using markers for pro-inflammatory M1 (iNOS, red) and anti-inflammatory M2 (CD206, green) phenotypes. In human AD tissues, iNOS expression was substantially increased, while CD206 was decreased, indicating a shift toward M1 polarization (Fig. 1 G). Quantification showed significantly higher iNOS intensity (Fig. 1 H) and lower CD206 intensity (Fig. 1 I) in AD tissues compared to controls.In mouse AD tissues, a similar M1-dominant polarization was evident, with elevated iNOS and reduced CD206 expression (Fig. 1 J). Relative fluorescence intensity was significantly increased for iNOS (Fig. 1 K) and decreased for CD206 (Fig. 1 L) in AD versus control tissues. These results indicate a critical role of M1 macrophages in AD pathogenesis. These findings highlight the pivotal contribution of M1 macrophages to AD development, suggesting a substantial accumulation of both M1 cells and exosomes in AD tissues from both humans and mice, with close spatial association between macrophages and exosomes. This suggests that M1 macrophages may contribute to AD progression through exosome-mediated intercellular communication. 3.2. Inhibition of exosome secretion by GW4869 attenuates BAPN-induced aortic dissection progression in mice A mouse model of AD was established by administering β-Aminopropionitrile (BAPN) to induce disease, followed by treatment with the exosome secretion inhibitor GW486932[ 31 ] or DMSO (vehicle) every 48 hours via intraperitoneal injection for 4 weeks (Fig. 2 A). BAPN administration significantly increased mortality compared to saline controls, with survival curves showing a marked decline in the BAPN+DMSO group (Fig. 2 B). Treatment with GW4869 significantly improved survival in BAPN-treated mice (Fig. 2 B). Consistent with the survival data, mortality rates were 66.7% in the BAPN+DMSO group and reduced to 33.3% in the BAPN+GW4869 group (Fig. 2 D). Gross morphological examination at week 7 revealed severe aortic dilation, tortuosity, and frequent intramural hematoma/rupture in BAPN+DMSO-treated mice, whereas GW4869 treatment substantially preserved aortic architecture and reduced macroscopic lesions (Fig. 2 C). Quantification confirmed a significantly lower AD incidence in the BAPN+GW4869 group (46.7%) compared to BAPN+DMSO (86.7%; Fig. 2 E). Maximum aortic diameter was markedly increased in BAPN+DMSO mice but significantly attenuated by GW4869 treatment (Fig. 2 F). Histological analyses further confirmed that GW4869 alleviated structural changes caused by BAPN, including luminal expansion (H&E staining), elastic fiber fragmentation (EVG staining), and collagen deposition (Masson staining) (Fig. 2 G and H). These results indicate that inhibition of exosome secretion with GW4869 significantly mitigates BAPN-induced AD progression, including reduced mortality, AD incidence, aortic dilation, elastic fiber degradation, and structural remodeling. This suggests that macrophage-derived exosomes play a critical pathogenic role in AD development. 3.3. Characterization and purification of extracellular vesicles from macrophages To investigate the role of macrophage-derived exosomes in AD, M1 polarization of RAW264.7 cells was induced using 100 ng/mL LPS and 20 ng/mL IFN-γ. Purification of exosomes from conditioned medium of M0 (M0-Exos) and M1 (M1-Exos) macrophages was performed after polarization. At the optimal concentrations of 100 ng/mL LPS and 20 ng/mL IFN-γ, the expression of iNOS, a marker specific to M1, was significantly increased (Fig. 3 A and C). Additionally, the macrophages underwent noticeable morphological alterations, with multipolar extensions, in contrast to the smaller and spherical control macrophages (Fig. 3 E). WB confirmed successful M1 polarization (Fig. 3 B and D). Exosomes were isolated via ultracentrifugation, displaying high expression of exosomal markers (TSG101, CD9, CD63) without contamination by endoplasmic reticulum(ER) marker Calnexin or cytoplasmic marker GAPDH (Fig. 3 F). Transmission electron microscopy showed exosomes had typical bilayer membranes and a mean diameter centering around 100 nm (Fig. 3 G). Nanoparticle tracking analysis corroborated this, with both M0-Exos and M1-Exos exhibiting size distributions peaking at approximately 100 nm (Fig. 3 H and I). Collectively, these characterizations validate the successful generation of M1-polarized macrophages and isolation of high-purity exosomes, setting the foundation for evaluating their functional impact on AD progression. 3.4. Exosomes derived from M1 macrophages promote phenotypic transition of vascular smooth muscle cells (VSMCs) In the pathogenesis of AD, intercellular crosstalk between macrophages and VSMCs plays a pivotal role, with exosomes potentially serving as key mediators, and VSMC phenotypic switching from contractile to synthetic states being essential for disease initiation and progression[ 12 , 32 – 35 ]. To determine whether M1 macrophage-derived exosomes influence phenotypic switching in VSMCs, primary mouse aortic VSMCs were exposed to exosomes isolated from M0- or M1-polarized RAW264.7 cells. Initially, primary VSMCs (Fig. 4 A) were incubated with PKH26-labeled M1-Exos or control M0-Exos for 24 hours. Cellular uptake of PKH26-labeled exosomes (red) by VSMCs was visualized using confocal microscopy, with VSMCs labeled for F-actin (green) and nuclei (blue) (Fig. 4 B). Protein-level assessment by Western blotting revealed diminished expression of contractile markers (MYOCD, α-SMA, CNN, SM22α) in M1-Exos-treated VSMCs compared to M0-Exos (Fig. 4 C), with densitometric quantification confirming statistically significant decreases (Fig. 4 D). Transcriptional profiling via qRT-PCR similarly showed suppressed mRNA levels for MYOCD, SM22α, α-SMA, and CNN in the M1-Exos group (Fig. 4 E–H) To verify exosome-specific effects, a non-contact co-culture system using RAW264.7 macrophages (upper chamber) and VSMCs (lower chamber) was established (Fig. 4 I). LPS/IFN-γ-polarized M1 macrophages markedly reduced the expression of VSMCs contractile markers (Fig. 4 J-N). However, addition of the exosome secretion inhibitor GW4869 (20 µM) partially restored contractile marker expression without altering M0 baselines (Fig. 4 J-N). Together, this highlights a critical mechanism whereby M1-Exos facilitate VSMC transdifferentiation. This exosome-mediated crosstalk is pivotal in AD pathogenesis, implicating it as a potential therapeutic target. 3.5. M1-derived exosomes exacerbate BAPN-induced AD progression and promote phenotypic switching in VSMCs To further confirm the functional roles of macrophage-derived exosomes in AD, exosomes secreted by RAW264.7 macrophages polarized into either the M1 (M1-Exos) or M0 (M0-Exos) states were isolated and characterized. An AD model was subsequently induced in mice via BAPN administration, followed by intravenous injection of either M1-Exos or control M0-Exos (20µg protein/150 µl) every 48h starting at week 3 until termination at week 7 (Fig. 5 A). M1-Exos treatment significantly increased mortality (Fig. 5 D), accelerated aortic rupture (Fig. 5 B), and intensified aortic dilation (Fig. 5 C and F) compared with the M0-Exos group. Morphometric analysis confirmed an increased maximum aortic diameter in mice treated with M1-Exos (Fig. 5 C and F) and higher incidence of AD (Fig. 5 E). Tissue-level examination via H&E staining uncovered extensive cavity enlargement and wall thinning in BAPN+M1-Exos sections, while EVG staining exposed severe elastic lamina disruption (arrows marking fractures) and Masson's trichrome indicated heightened fibrosis (Fig. 5 H). Molecular profiling of aortic extracts by Western blotting showed reduced levels of contractile indicators (MYOCD, α-SMA, CNN, SM22α) in BAPN+M1-Exos tissues (Fig. 5 I), with densitometry confirming significant declines (Fig. 5 J). These results indicate that M1-Exos intensify AD severity in vivo, amplifying structural damage and VSMC dedifferentiation. 3.6. M1-derived exosomes induce phenotypic switching of VSMCs through miR-155-5p delivery Exosomes mediate intercellular communication by transporting bioactive cargo such as miRNAs and proteins, influencing cellular signaling pathways and functions, thereby contributing to disease pathogenesis[ 36 ]. Given previous findings that M1-Exos exacerbate AD, we sought to identify the key functional exosomal components responsible for this effect. we conducted miRNA sequencing on exosomes from M1- and M0-polarized RAW264.7 macrophages, complemented by analysis of public datasets. Heatmap visualization based on sequencing data of exosomes derived from M0 and M1 macrophages showed the upregulation of specific miRNAs in M1 cells, including a prominent increase in miR-155-5p (Fig. 6 A). Volcano plots from our exosomal sequencing highlighted differentially expressed miRNAs, with miR-155-5p markedly upregulated in M1-Exos ( Fig. S1 A ). Integration with GSE125171 (BMDMs) revealed consistent patterns: heatmaps and volcano plots showed miR-155-5p among top upregulated miRNAs in M1-BMDMs (Fig. S1 B and C). Our cellular sequencing of RAW264.7 similarly identified miR-155-5p enrichment in M1 states via heatmaps and volcano plots (Fig. S1 D and E). A Venn diagram cross-referencing predicted targets, GSE125171 differentials, and our exosomal miRNAs pinpointed miR-155-5p as the overlapping hit (Fig. 6 B). Therefore, we hypothesized that M1-Exos accelerate AD by transferring miR-155-5p to VSMCs and inducing a synthetic phenotype. To test this hypothesis, miR-155-5p expression was first verified by qRT-PCR, which showed significant upregulation in M1 macrophages stimulated by LPS/IFN-γ and their exosomes (Fig. 6 C and D). Elevated miR-155-5p expression was confirmed in human and murine AD tissues compared to normal controls (Fig. 6 E and F). RNA-FISH further revealed notable accumulation of miR-155-5p in AD tissues, consistent with sequencing data (Fig. 6 G and H). Next, M1 macrophages were transfected with FAM-labeled miR-155-5p mimics, and exosomes isolated from conditioned media were incubated with VSMCs. Delivery experiments confirmed exosomal shuttling: FAM-miR-155-5p (green) overlapped with PKH26-Exos (red) in VSMC cytoplasm (Fig. 6 I). Additionally, qRT-PCR confirmed elevated miR-155-5p levels within treated VSMCs (Fig. 6 J). Pretreatment of exosomes with Triton X-100, which disrupts membranes prior to RNase digestion, significantly reduced miR-155-5p levels transferred into VSMCs. However, RNase treatment alone had little effect (Fig. 6 K). These results suggest that exosomal packaging protects miR-155-5p from degradation, facilitating functional delivery. Exosomes (miR-155 OE -Exos) were extracted from RAW264.7 macrophages that had been engineered to stably express high levels of miR-155-5p (miR-155 OE ), in order to evaluate the functional significance of miR-155-5p. The results of the qRT-PCR assay confirmed the increased expression of miR-155-5p in both cells and exosomes (Fig. 6 L and M). WB examination (Fig. 6 N and R) indicated that incubating VSMCs with miR-155 OE -Exos significantly reduced levels of contractile phenotypic markers such as α-SMA, SM22α, CNN, and MYOCD, which is similar to the effect seen with exosomes produced from M1. These findings demonstrate that M1-derived exosomes promote VSMC phenotype switching through miR-155-5p transfer, contributing to AD progression. 3.7. miR-155-5p negatively regulates SMAD5 expression to control VSMC phenotypic switching To uncover the downstream mechanism by which exosomal miR-155-5p induces VSMC dedifferentiation, we integrated RNA-seq data from treated VSMCs with miRNA target prediction databases (miRDB and TargetScan). Venn analysis revealed 17 overlapping differentially expressed mRNAs (Fig. 7 A). Network mapping prioritized SMAD5 as a high-confidence target with strong connectivity (Fig. 7 B). Enrichment analysis showed these candidates were involved in transcriptional regulation, protein phosphorylation, angiogenesis, and morphological transformation, relevant to AD pathogenesis (Fig.S2A-C). GO enrichment analysis of DEGs in miR-155 OE -Exos-treated VSMCs revealed significant overrepresentation of terms related to transcription factor activity, DNA-binding regulation, and cell homeostasis (Fig. S3A), further supporting a role in transcriptional reprogramming during phenotypic switching. We next screened 17 candidate target genes in primary VSMCs exposed to miR-155 OE -Exos versus miR-155 NC -Exos using qRT-PCR. Among them, E2F2, SMAD5, STAB1, PICLAM, KDM3A and SDCBP showed consistent and significant downregulation (Fig. S3N-S), whereas other candidates (HIF1A, ITK, TAB2, GNAS, etc.) exhibited no significant or inconsistent changes (Fig. S3C–M). Tissue-level validation confirmed these findings: qRT-PCR screening of candidate genes (E2F2, SMAD5, STAB1, PICLAM, KDM3A, SDCBP) in both human and mouse AD aortic tissues (Fig. S4A). In human samples, SMAD5, STAB1, E2F2 and PICLAM mRNA levels were significantly reduced in AD compared to controls (Fig. S4B–e), whereas KDM3A and SDCBP showed no consistent change (Fig. S4F and G). Similar downregulation of SMAD5, STAB1, and E2F2 was observed in mouse AD tissues (Fig. S4H–J), with KDM3A, PICLAM and SDCBP unchanged (Fig. S4L–M). WB screening of candidate genes (E2F2, SMAD5, STAB1) in both human and mouse AD aortic tissues (Fig. S5A). In mouse samples, SMAD5 and STAB1 protein levels were significantly reduced in AD compared to controls (Fig. S5B-D), whereas E2F2 showed no consistent change (Fig. S5E). Similar downregulation of SMAD5 was observed in human AD tissues (Fig. S5F and G), with E2F2 and STAB1 unchanged (Fig. S5H and I). Validation pipeline combined qRT-PCR for mRNA candidates in treated VSMCs and AD tissues, followed by protein confirmation via WB in human and mouse AD samples (Fig. 7 C). Among candidates, SMAD5 showed consistent downregulation. IF staining further corroborated reduced SMAD5 and SM22α expression in AD aortic walls from humans and mice, with diminished colocalization in merged images (Fig. 7 D and E). Quantitative fluorescence analysis confirmed significant downregulation of both proteins in AD tissues (Fig. 7 F-I). TargetScan bioinformatic predictions and dual-luciferase assays provided evidence confirming direct interaction of miR-155-5p with the SMAD5 3′-UTR region (Fig. 7 J, K). To further establish the functional consequence of this interaction in the context of exosomal delivery, primary VSMCs were treated with exosomes derived from miR-155-overexpressing RAW264.7 cells (miR-155 OE -Exos). Immunoblot analysis further demonstrated reduced SMAD5 protein levels upon miR-155-5p upregulation (Fig. 7 L and M). Rescue experiments indicated SMAD5 overexpression in miR-155-5p-treated VSMCs restored expression of contractile markers (α-SMA, SM22α, CNN, MYOCD) suppressed by miR-155-5p (Fig. 7 N–S). Collectively, these results demonstrate that exosomal miR-155-5p targets SMAD5 to induce synthetic phenotypic switching in VSMCs, thereby contributing to AD progression. 3.8. Engineered Antago-miR-155-5p exosomes suppress AD progression by restoring SMAD5 and contractile phenotype Our results demonstrated that exosomal miR-155-5p from M1 macrophages promoted BAPN-induced AD through VSMC phenotypic switching by targeting SMAD5. Given previous reports of elevated serum miR-155-5p in AD patients, suggesting its potential as a diagnostic biomarker [55], we hypothesized that exosomal miR-155-5p promotes AD via regulation of SMAD5 and VSMC phenotype. To evaluate these findings in vivo, mice were subjected to BAPN-induced AD modeling. Animals were randomized into two experimental cohorts (n = 15/group) receiving intravenous administration of exosomes loaded either with Antago-miR-155-5p (Antago-miR-155-5p-Exos) or corresponding negative controls (Antago-miR-NC-Exos) (Fig. 8 A). Antago-miR-155-5p-Exos significantly reduced mortality (Fig. 8 D), delayed aortic rupture (Fig. 8 B), and decreased aortic dilation (Fig. 8 C, F). Anatomical and histological assessments showed reduced AD incidence (Fig. 8 E) and mitigated BAPN-induced structural damage, including luminal dilation, elastin fragmentation, and collagen deposition (H&E, EVG, Masson staining) (Fig. 8 G, H). At the molecular level,WB analysis indicated that Antago-miR-155-5p-Exos reversed reductions in contractile markers (MYOCD, α-SMA, SM22α, CNN) and restored SMAD5 expression (Fig. 8 I, J). IF confirmed increased SMAD5 (green) and SM22α (red) fluorescence intensity and stronger colocalization in the media of Antago-miR-155-5p-Exos-treated aortas (Fig. 8 K). Quantitative analysis of fluorescence intensity verified significant restoration of both proteins (Fig. S6A and B). These findings indicate that inhibiting exosomal miR-155-5p ameliorates BAPN-induced AD progression by restoring SMAD5 expression, maintaining the VSMC contractile phenotype, and attenuating vascular remodeling and structural damage. SMAD5 thus emerges as a critical downstream effector of miR-155-5p in AD pathogenesis. 4. Discussion In this study, we identified that M1 macrophage-derived exosomal miR-155-5p exacerbates aortic dissection (AD) progression by targeting SMAD5, thereby promoting vascular smooth muscle cell (VSMC) phenotypic switching from a contractile to a synthetic state. Notably, engineered exosomes loaded with Antago-miR-155-5p effectively reversed this pathological process, significantly reducing AD incidence, mortality, and vascular remodeling in the BAPN-induced mouse model. These findings uncover a novel macrophage-VSMC communication axis in AD pathogenesis and provide strong preclinical evidence supporting exosome-based miRNA-targeted therapy as a promising strategy for aortic dissection. A growing body of evidence underscores inflammation and infiltration of immune cells as critical mechanisms in the progression of AD[ 37 – 39 ]. Early-stage activation and recruitment of immune cells into the aortic wall are crucial initiating steps in AD pathogenesis[ 40 ]. According to Xue et al., macrophages and monocytes serve as initial responders, facilitating subsequent infiltration by neutrophils and T lymphocytes into the vascular wall[ 41 ]. Macrophage accumulation strongly correlates with vascular damage and rupture38. However, AD involves complex interactions among diverse cell populations. Traditionally, macrophage–VSMC communication was attributed primarily to cytokines[ 16 ]. Recent research identifies exosomes as essential mediators of cell-to-cell communication through delivery of bioactive molecules[ 42 , 43 ]. Our study further demonstrates that M1 macrophages regulate VSMC phenotype transition through exosome-mediated transfer of miR-155-5p. Exosomes, as natural carriers, enable precise intercellular regulation and show increasing promise in disease mechanisms and therapeutics[ 44 ]. Using multi-omics approaches and functional validation, miR-155-5p emerged as a central exosomal effector driving VSMCs toward a synthetic phenotype via suppression of SMAD5, consequently promoting AD. SMAD5, a key mediator of the BMP/TGF-β signaling axis, is essential for maintaining VSMC contractile phenotype and vascular homeostasis[ 45 , 46 ]. Our demonstration that miR-155-5p directly suppresses SMAD5 expression provides a molecular link between inflammatory exosomal signaling and VSMC dedifferentiation in AD, revealing a novel upstream regulatory node that may be amenable to therapeutic targeting. Leveraging the natural biocompatibility, low immunogenicity, and homing potential of macrophage-derived exosomes[ 47 – 49 ], we engineered an Antago-miR-155-5p delivery system that significantly ameliorated AD progression in vivo. This approach offers advantages over synthetic nanoparticles, including enhanced stability of RNA cargo and potential for endogenous cell-specific targeting, positioning exosome-mediated miRNA inhibition as a viable strategy for future AD therapeutics. However, several limitations exist. First, the in vitro M1 polarization protocol (LPS/IFN-γ) may not fully recapitulate the heterogeneous inflammatory milieu in vivo. Second, while Antago-miR-155-5p-loaded exosomes showed robust efficacy, their long-term safety, biodistribution, and potential immunogenicity require further evaluation in larger preclinical models. Future studies using cell-specific miR-155 knockout mice and conditional SMAD5 ablation will be essential to delineate contribution and specificity. In conclusion, our study identifies and characterizes the M1 macrophage-exosome-miR-155-5p-SMAD5 axis as a critical regulator of VSMC phenotypic switching in AD. These findings improve the mechanistic understanding of AD and provide new therapeutic targets. Furthermore, this research strongly supports the clinical development of exosome-based gene therapy approaches 5. Conclusion In this study, we identified that M1 macrophage-derived exosomal miR-155-5p exacerbates AD progression by targeting SMAD5, thereby promoting VSMC phenotypic switching from a contractile to a synthetic state. Notably, engineered exosomes loaded with Antago-miR-155-5p effectively reversed this pathological process, significantly reducing AD incidence, mortality, and vascular remodeling in the BAPN-induced mouse model. These findings uncover a novel macrophage-VSMC communication axis in AD pathogenesis and provide strong preclinical evidence supporting exosome-based miRNA-targeted therapy as a promising strategy for aortic dissection. Declarations Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements Dataset supporting the current research results can be obtained from the corresponding author on justifiable request. Author Contributions Dengwei Cao and Xinyi Li performed all experiments, data analysis and drafted the manuscript. Shaoping Zhu, Jianfeng Chen and Haoxiang Li assisted with in vitro experiment. Jiajun Shi, Xiaoqi Xiong, and Yumou Wang assisted with in vivo experiment. Zhe Dong assisted with human aortic tissue samples collection and reviewed the manuscript. Jinping Liu, Xinyi Li and Zhe Dong provided financial support, research design, manuscript revision, and final manuscript approval. Funding Financial support for this study was provided by the National Natural Science Foundation of China (No.82470421 to LJP; No.82472204 to LXY), Funding was also received from the Central Universities’ Fundamental Research Funds (2042024kf0025 to LJP),the Hubei Provincial Key Research and Development Project (2023BCB002 to LJP), additional grants were provided by the Hubei Provincial Natural Science Foundation (No.2022CFC026 to DZ), and the Hubei Province Health Commission(No.WJ2023M061 to DZ Data Availability Statement The data that support the findings of this study are available from the corresponding author upon legitimate inquiry. Conflict of Interest The authors state no conflicts of interest. 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Oncel, Exosomes: Large-scale production, isolation, drug loading efficiency, and biodistribution and uptake, Journal of Controlled Release : Official Journal of the Controlled Release Society 347 (2022) 533-543. Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8986811","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":600101400,"identity":"54874817-acfe-469d-b460-ddbb2c5c54fd","order_by":0,"name":"Dengwei Cao","email":"","orcid":"","institution":"Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Dengwei","middleName":"","lastName":"Cao","suffix":""},{"id":600101401,"identity":"d1c93981-ba5b-4a67-9b51-d7f0ff676a01","order_by":1,"name":"Xinyi Li","email":"","orcid":"","institution":"Wuhan 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10:26:02","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8986811/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8986811/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103990940,"identity":"18a06612-d3f2-41cd-94a4-02ddc5291eb7","added_by":"auto","created_at":"2026-03-05 11:37:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11033417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignificant accumulation of M1 macrophages and exosomes in Aortic Dissection (AD) tissues\u003c/strong\u003e \u003cstrong\u003efrom humans and mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative immunofluorescence (IF) images of human aortic tissues stained for the exosomal marker TSG101 (green), macrophage marker CD68 (red), and nuclear counterstain DAPI (blue), with merged overlay. Scale bar = 50 μm. \u003cstrong\u003eB.\u003c/strong\u003e Quantification of relative fluorescence intensity for TSG101 in human aortic tissues (Control vs. AD). \u003cstrong\u003eC.\u003c/strong\u003eQuantification of relative fluorescence intensity for CD68 in human aortic tissues (Control vs. AD). \u003cstrong\u003eD.\u003c/strong\u003e Representative IF images of mouse aortic tissues stained for TSG101 (green), macrophage marker F4/80 (red), and DAPI (blue), with merged overlay. Scale bar = 20 μm. \u003cstrong\u003eE. \u003c/strong\u003eQuantification of relative fluorescence intensity for TSG101 in mouse aortic tissues (Control vs. AD). \u003cstrong\u003eF. \u003c/strong\u003eQuantification of relative fluorescence intensity for F4/80 in mouse aortic tissues (Control vs. AD). \u003cstrong\u003eG.\u003c/strong\u003eRepresentative IF images of human aortic tissues stained for M1 macrophage marker iNOS (green), M2 macrophage marker CD206 (red), and DAPI (blue), with merged overlay. Scale bar = 50 μm. \u003cstrong\u003eH.\u003c/strong\u003e Quantification of relative fluorescence intensity for iNOS in human aortic tissues (Control vs. AD). \u003cstrong\u003eI. \u003c/strong\u003eQuantification of relative fluorescence intensity for CD206 in human aortic tissues (Control vs. AD). \u003cstrong\u003eJ.\u003c/strong\u003e Representative IF images of mouse aortic tissues stained for iNOS (green), CD206 (red), and DAPI (blue), with merged overlay. Scale bar = 50 μm.\u003cstrong\u003eK. \u003c/strong\u003eQuantification of relative fluorescence intensity for iNOS in mouse aortic tissues (Control vs. AD). \u003cstrong\u003eL.\u003c/strong\u003eQuantification of relative fluorescence intensity for CD206 in mouse aortic tissues (Control vs. AD). Mean ± SEM. **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001by unpaired Student's t-test (two-tailed).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/8bf0c5ca2a33d7262d729ab7.png"},{"id":103990949,"identity":"2d1df63b-998f-4704-9da6-3fdee6ce5c71","added_by":"auto","created_at":"2026-03-05 11:37:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16337162,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGW4869-mediated inhibition of exosome release attenuates AD progression in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA．\u003c/strong\u003e Schematic timeline of the experimental design. Three-week-old male mice were administered BAPN (1 g/kg/day) or saline in drinking water starting at week 3. GW4869 (exosome secretion inhibitor) or DMSO (vehicle) was administered intraperitoneally every 48 h from week 3 until week 7 (end of experiment). \u003cstrong\u003eB. \u003c/strong\u003eSurvival analysis among the experimental groups (n = 15 per group). \u003cstrong\u003eC. \u003c/strong\u003eRepresentative gross morphological images of the aorta (Scale bar: 1 cm). \u003cstrong\u003eD.\u003c/strong\u003eQuantification of mortality rates across groups (n = 15 per group). E. Incidence rates of AD within experimental groups (n = 15 per group). \u003cstrong\u003eF. \u003c/strong\u003eMaximum aortic diameter measurements normalized by body weight (n = 6 per group). \u003cstrong\u003eG.\u003c/strong\u003eScoring of elastin degradation severity (n = 6 per group).\u003cstrong\u003e H. \u003c/strong\u003eRepresentative histological images of aortic sections stained with H\u0026amp;E, EVG, and Masson techniques (day 28 post-BAPN). black arrows highlight fragmented elastin fibers. Mean ± SEM. The survival curves in \u003cstrong\u003eB\u003c/strong\u003e were analyzed using the Kaplan-Meier curve and compared using the log-rank test. Data in \u003cstrong\u003eF and G\u003c/strong\u003e was analyzedusing a two-way ANOVA followed by Tukey post hoc test. *P \u0026lt; 0.05; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/25b4db98baad34518bac5898.png"},{"id":103990944,"identity":"ba9efa36-4e4d-4c7c-8403-862095ddeae8","added_by":"auto","created_at":"2026-03-05 11:37:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11353697,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePolarization of RAW264.7 macrophages to M1 phenotype and characterization of derived exosomes.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Protein expression levels of iNOS in macrophages exposed to LPS (100 ng/mL) in combination with incremental IFN-γ concentrations (2.5, 5, 10, and 20 ng/mL). \u003cstrong\u003eB.\u003c/strong\u003eWB analysis evaluating iNOS expression in macrophages treated with LPS (100 ng/mL) and IFN-γ (20 ng/mL). \u003cstrong\u003eC. \u003c/strong\u003eQuantitative immunoblot analysis of iNOS protein expression from experiments shown in panel \u003cstrong\u003eA\u003c/strong\u003e. \u003cstrong\u003eD.\u003c/strong\u003eCorresponding quantification of iNOS protein levels derived from immunoblots in panel \u003cstrong\u003eB\u003c/strong\u003e. \u003cstrong\u003eE.\u003c/strong\u003e Representative images demonstrating macrophage morphology after polarization toward the M1 state induced by LPS and IFN-γ stimulation (scale bar: 100 μm). \u003cstrong\u003eF. \u003c/strong\u003eImmunoblot validation of isolated exosomes showing positive markers (CD9, CD63, TSG101) and negative controls (GAPDH, Calnexin). \u003cstrong\u003eG.\u003c/strong\u003e Transmission electron microscopy (TEM) images of M0-Exos and M1-Exos at magnifications of 500 nm, 200 nm, and 100 nm. Arrows indicate typical cup-shaped morphology with bilayer membranes. \u003cstrong\u003eH.\u003c/strong\u003eNanoparticle tracking analysis (NTA) demonstrating M0-Exos size distribution (average peak size: 92 ± 5 nm).\u003cstrong\u003e I.\u003c/strong\u003e Nanoparticle tracking analysis (NTA) demonstrating M1-Exos size distribution (average peak size: 92 ± 5 nm). Mean ± SEM. Data in \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD \u003c/strong\u003ewere analyzed via unpaired Student \u003cem\u003et\u003c/em\u003e test. *P \u0026lt; 0.05, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/b077d4950890a4c9ac00927d.png"},{"id":103990943,"identity":"09c52993-ba97-40e3-84fc-1298a2652fac","added_by":"auto","created_at":"2026-03-05 11:37:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8293177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosomes from M1 macrophages induce vascular smooth muscle cells (VSMCs) phenotypic switching.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eIF characterization of primary mouse VSMCs. Scale bar = 25 μm. \u003cstrong\u003eB. \u003c/strong\u003eExosomes were labeled with PKH26 (red) and co-incubated with VSMCs stained for F-actin (488-phalloidin, green) and nuclei (Hoechst 33342, blue). \u003cstrong\u003eC.\u003c/strong\u003e WB analysis of contractile marker expression (MYOCD, α-SMA, CNN, SM22α) in VSMCs after treatment with M1-Exos or M0-Exos. \u003cstrong\u003eD.\u003c/strong\u003e Quantification of relative protein expression levels from panel \u003cstrong\u003eC\u003c/strong\u003e. \u003cstrong\u003eE.\u003c/strong\u003e Relative mRNA expression of MYOCD in VSMCs treated with M0-Exos or M1-Exos, assessed by qRT-PCR.\u003cstrong\u003eF. \u003c/strong\u003eRelative mRNA expression of SM22α in VSMCs treated with M0-Exos or M1-Exos.\u003cstrong\u003eG.\u003c/strong\u003e Relative mRNA expression of α-SMA in VSMCs treated with M0-Exos or M1-Exos.\u003cstrong\u003eH.\u003c/strong\u003e Relative mRNA expression of CNN in VSMCs treated with M0-Exos or M1-Exos. \u003cstrong\u003eI. \u003c/strong\u003eSchematic of the non-contact co-culture system using Transwell inserts, with M0- or M1-polarized RAW264.7 macrophages in the upper chamber and VSMCs in the lower chamber, with or without GW4869 pretreatment. \u003cstrong\u003eJ. \u003c/strong\u003eWB assessment of contractile markers in VSMCs after 24-hour co-culture. K. Quantification of relative MYOCD protein expression (normalized to GAPDH) from panel \u003cstrong\u003eJ\u003c/strong\u003e.\u003cstrong\u003eL. \u003c/strong\u003eQuantification of relative α-SMA protein expression from panel \u003cstrong\u003eJ\u003c/strong\u003e.\u003cstrong\u003eM\u003c/strong\u003e. Quantification of relative CNN protein expression from panel \u003cstrong\u003eJ\u003c/strong\u003e.\u003cstrong\u003eN\u003c/strong\u003e. Quantification of relative SM22α protein expression from panel \u003cstrong\u003eJ\u003c/strong\u003e. Data in \u003cstrong\u003eD through H\u003c/strong\u003e was analyzedusing unpaired Student's t-test .Data in \u003cstrong\u003eK through N\u003c/strong\u003e was analyzedusing a two-way ANOVA followed by Tukey post hoc test. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/3e2ee79172e30863fe98e647.png"},{"id":104779317,"identity":"ae4a4f09-0132-4b97-9c87-b6ab768af5da","added_by":"auto","created_at":"2026-03-17 07:38:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":17385190,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosomes from M1 macrophages exacerbate BAPN-induced AD progression in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA．\u003c/strong\u003e Schematic of the experimental timeline. saline in drinking water from week3. M1-Exos or M0-Exos (tail vein injection, 150μl/20 μg protein) were administered every 48 h until week 7. \u003cstrong\u003eB.\u003c/strong\u003e Survival analysis among the experimental groups (n = 15 per group). \u003cstrong\u003eC.\u003c/strong\u003e Representative macroscopic images of whole aortas (scale bar: 1 cm). \u003cstrong\u003eD.\u003c/strong\u003e Mortality analysis among experimental mouse groups (n = 15 per group). \u003cstrong\u003eE.\u003c/strong\u003e Comparison of AD occurrence rates across experimental groups (n = 15 per group). \u003cstrong\u003eF. \u003c/strong\u003eQuantitative assessment of maximum aortic diameters normalized to body weight (n = 6 per group). \u003cstrong\u003eG.\u003c/strong\u003e Assessment of elastin fiber damage severity within aortic tissues (n = 6 per group). \u003cstrong\u003eH. \u003c/strong\u003eHistopathological assessment of aortic sections via H\u0026amp;E, EVG, and Masson staining methods at day 28 post-BAPN administration; Upper rows: Low-magnification views (scale bar = 200 μm); lower rows: High-magnification details (scale bar = 20 μm). Black arrows in EVG panels denote elastic fiber breaks.arrows denote regions of disrupted elastin fibers. \u003cstrong\u003eI. \u003c/strong\u003eImmunoblot detection of contractile phenotype-associated proteins (MYOCD, α-SMA, SM22α, CNN) in aortic tissues following exosome intervention.\u003cstrong\u003e J. \u003c/strong\u003eDensitometric quantification of WB results displayed in panel\u003cstrong\u003e I\u003c/strong\u003e. Mean ± SEM. The survival curves in \u003cstrong\u003eB\u003c/strong\u003e were analyzed using the Kaplan-Meier curve and compared using the log-rank test. Data in\u003cstrong\u003e F\u003c/strong\u003e,\u003cstrong\u003eG\u003c/strong\u003e and \u003cstrong\u003eJ\u003c/strong\u003e was analyzed via unpaired Student t test.*P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/0c715f2d55604db254e72206.png"},{"id":104402412,"identity":"6a16fe32-49a2-4d41-ae56-d58bdf2dc504","added_by":"auto","created_at":"2026-03-11 12:15:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9401963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExosomal miR-155-5p secreted by M1 macrophages facilitates phenotypic transformation of VSMCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Sequencing-based heatmap illustrating significantly altered miRNAs within exosomes. \u003cstrong\u003eB.\u003c/strong\u003e Venn diagram showing intersections of differentially expressed miRNAs among RAW264.7 macrophages, their secreted exosomes, and publicly available BMDM data (GSE125171). \u003cstrong\u003eC.\u003c/strong\u003e Expression levels of miR-155-5p measured by qRT-PCR in RAW264.7 cells polarized into M0 or M1 states. \u003cstrong\u003eD.\u003c/strong\u003e qRT-PCR quantification of miR-155-5p in exosomes isolated from M0- and M1-polarized macrophages. \u003cstrong\u003eE.\u003c/strong\u003e Relative miR-155-5p expression in mouse aortic tissues (Control vs. AD), assessed by qRT-PCR. \u003cstrong\u003eF. \u003c/strong\u003eRelative miR-155-5p expression in human aortic tissues (Control vs. AD), assessed by qRT-PCR. \u003cstrong\u003eG.\u003c/strong\u003e RNA fluorescence in situ hybridization (RNA-FISH) images of miR-155-5p (red) in mouse aortic tissues (Control vs. AD). Scale bar = 20 μm. \u003cstrong\u003eH.\u003c/strong\u003e RNA-FISH images of miR-155-5p (red) in human aortic tissues (Control vs. AD). Scale bar = 50 μm. \u003cstrong\u003eI.\u003c/strong\u003e Confocal images showing transfer of FAM-labeled miR-155-5p (green) from PKH26-labeled exosomes (red) to VSMCs, with merged overlay and Hoechst nuclear stain (blue). Scale bar = 25 μm. \u003cstrong\u003eJ.\u003c/strong\u003e miR-155-5p expression in VSMCs following exposure to exosomes from M0 or M1 macrophages measured by qRT-PCR. \u003cstrong\u003eK. \u003c/strong\u003eqRT-PCR analysis of exosomal miR-155-5p after RNase digestion with or without Triton X-100 permeabilization. \u003cstrong\u003eL.\u003c/strong\u003e qRT-PCR confirming miR-155-5p overexpression in RAW264.7 cells transfected with miR-155 mimic (miR-155\u003csup\u003eOE\u003c/sup\u003e) versus negative control (miR-NC\u003csup\u003eOE\u003c/sup\u003e). \u003cstrong\u003eM.\u003c/strong\u003e qRT-PCR of miR-155-5p in exosomes derived from miR-NC\u003csup\u003eOE\u003c/sup\u003e- or miR-155\u003csup\u003eOE\u003c/sup\u003e-RAW264.7 cells. \u003cstrong\u003eN.\u003c/strong\u003e Immunoblot analysis of contractile-related proteins (MYOCD, α-SMA, SM22α, CNN) in VSMCs. \u003cstrong\u003eO-R. \u003c/strong\u003eQuantitative analysis corresponding to WB data in panel (N). Mean ± SEM. Data \u003cstrong\u003eK,O,P,Q and R\u003c/strong\u003e was analyzed using a two-way ANOVA followed by Tukey post hoc test. Data from other experiments were analyzed using an unpaired Student's t-test. **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/19a11e440e7e05e305d96420.png"},{"id":104402629,"identity":"319b96a6-db35-468b-858c-3fd2648df04a","added_by":"auto","created_at":"2026-03-11 12:15:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":16759284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSMAD5 is directly targeted by miR-155-5p and mediates VSMC phenotypic transition in AD.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Venn diagram illustrating the overlap of predicted miR-155-5p target genes (from miRDB and TargetScan databases) with downregulated genes identified by RNA sequencing (RNA-seq) of aortic tissues from BAPN-induced AD mice. \u003cstrong\u003eB.\u003c/strong\u003e List of the 17 candidate genes identified from the intersection shown in panel A.\u003cstrong\u003e C.\u003c/strong\u003e Workflow schematic for identifying miR-155-5p targets in AD. \u003cstrong\u003eD. \u003c/strong\u003eRepresentative IF images showing co-localization of SMAD5 (green) with the VSMC marker SM22α (red) in human aortic tissues from control and AD groups. Nuclei are stained with DAPI (blue). Scale bar: 50 μm. \u003cstrong\u003eE.\u003c/strong\u003e Representative IF of SMAD5 (green) and SM22α (red) in mouse aortic tissues. \u003cstrong\u003eF-G.\u003c/strong\u003e Quantitative fluorescence analysis from images shown in\u003cstrong\u003e E\u003c/strong\u003e. \u003cstrong\u003eH-I.\u003c/strong\u003e Quantitative fluorescence analysis from images shown in \u003cstrong\u003eD\u003c/strong\u003e. \u003cstrong\u003eJ.\u003c/strong\u003e Schematic of the miR-155-5p binding site in the SMAD5 3'-UTR (wild-type and mutant sequences used for luciferase reporter assays). \u003cstrong\u003eK.\u003c/strong\u003e Dual-luciferase reporter assay in VSMCs co-transfected with miR-155-5p mimic or negative control and SMAD5 3'-UTR wild-type (WT) or mutant (Mut) constructs. \u003cstrong\u003eL.\u003c/strong\u003e WB of SMAD5 protein expression in VSMCs after treatment with miR-155\u003csup\u003eOE\u003c/sup\u003e-Exos or controls. \u003cstrong\u003eM.\u003c/strong\u003e Quantification of SMAD5 protein levels from panel \u003cstrong\u003eL\u003c/strong\u003e. \u003cstrong\u003eN.\u003c/strong\u003e Western blot analysis of contractile markers (MYOCD, α-SMA, CNN, SM22α), and SMAD5 in VSMCs transfected with empty vector or SMAD5 overexpression plasmid. \u003cstrong\u003eO-S. \u003c/strong\u003eQuantification of WB data from panel \u003cstrong\u003eN\u003c/strong\u003e. Mean ± SEM. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001; ns, not significant (unpaired Student's t-test or one-way ANOVA with Tukey's post hoc test).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/4afd5abcc76e8a2f0e3ba042.png"},{"id":104402769,"identity":"2afe6c65-5ace-46aa-8272-1e1bc6cd6ad5","added_by":"auto","created_at":"2026-03-11 12:16:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":16140779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic inhibition of exosomal miR-155-5p attenuates AD progression in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eExperimental design: mice orally treated with BAPN or saline and intravenously injected with Antago-miR-155-5p-Exos or controls every 48 hours. \u003cstrong\u003eB.\u003c/strong\u003eSurvival curves for BAPN-treated mice receiving Antago-miR-NC-Exos or Antago-miR-155-5p-Exos(n=15/group). \u003cstrong\u003eC. \u003c/strong\u003eRepresentative images of aortic morphology. Scale bar = 1 cm. \u003cstrong\u003eD.\u003c/strong\u003e Quantification of mortality rates in BAPN-treated groups (n=15/group). \u003cstrong\u003eE. \u003c/strong\u003eIncidence of AD in BAPN-treated groups (n=15/group). \u003cstrong\u003eF.\u003c/strong\u003e Maximum aortic diameter normalized to body weight (n=6/group). \u003cstrong\u003eG. \u003c/strong\u003eElastin degradation scoring (n=6/group). \u003cstrong\u003eH.\u003c/strong\u003e Representative H\u0026amp;E, EVG, and Masson staining (28 days post-BAPN treatment). Black arrows denote elastic fiber fragmentation. \u003cstrong\u003eI. \u003c/strong\u003eWB for contractile markers (MYOCD, α-SMA, SM22α, CNN) in mouse aortic tissues. \u003cstrong\u003eJ. \u003c/strong\u003eQuantification of WB from panel \u003cstrong\u003eI\u003c/strong\u003e. \u003cstrong\u003eK.\u003c/strong\u003e Representative IF images of SMAD5 (green) and SM22α (red) in aortic tissues. Scale bar = 50 μm. Data are mean ± SEM. The survival curves in\u003cstrong\u003e B\u003c/strong\u003e were analyzed using the Kaplan-Meier curve and compared using the log-rank test. Data from other experiments were analyzed using an unpaired Student's t-test. *P \u0026lt; 0.05, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 (unpaired Student's t-test).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/96b75b1aec7666b5dd1d061b.png"},{"id":104779288,"identity":"59a1dc70-f6fe-484a-af11-c67f64ba613e","added_by":"auto","created_at":"2026-03-17 07:38:12","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1512712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic model of M1 macrophage-derived exosomal miR-155-5p exacerbating aortic dissection via SMAD5 in VSMCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter uptake by VSMCs, these M1 macrophage-derived exosomes introduce miR-155-5p into the cytoplasmic compartment. Once internalized, miR-155-5p downregulates SMAD5 expression, thereby prompting VSMCs to undergo phenotypic transition from contractile to synthetic forms and subsequently accelerating AD progression. Conversely, exosomes carrying Antago-miR-155-5p effectively counteract miR-155-5p, restore SMAD5 levels, maintain VSMC contractility, and alleviate AD progression.\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/946c77ce3673c1fe1017ec18.png"},{"id":109249294,"identity":"ca0cb7fe-294a-44ea-bd00-fdd324521ea9","added_by":"auto","created_at":"2026-05-14 08:47:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":143228165,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/751872d7-32d6-440e-b281-b4c7dd59b24b.pdf"},{"id":104402381,"identity":"6ceaf6e1-51c8-4ed9-8111-0089470790d0","added_by":"auto","created_at":"2026-03-11 12:15:12","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1914368,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile.doc","url":"https://assets-eu.researchsquare.com/files/rs-8986811/v1/2e9543f06e9a450b72bc11b4.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"M1 Macrophage-Derived Exosomal miR-155-5p Exacerbates Aortic Dissection Progression via SMAD5-Mediated Regulation of Vascular Smooth Muscle Cell Phenotype","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAortic dissection (AD) represents a lethal vascular condition involving the architectural degradation of the aortic wall.[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While operative management persists as the cornerstone of clinical therapy, postoperative mortality is still high. Currently, effective non-surgical strategies for reducing mortality associated with AD are insufficient [\u003cspan additionalcitationids=\"CR4 CR5 CR6\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, elucidating the mechanisms underlying AD progression, discovering new therapeutic targets, and identifying biomarkers for early diagnosis are crucial for improving early intervention and personalized treatment approaches.\u003c/p\u003e \u003cp\u003eAmong the various pathological processes involved in AD, inflammation and vascular remodeling are considered central. Accumulating evidence highlights critical roles for inflammatory cell infiltration [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], extracellular matrix (ECM) degradation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and VSMC phenotypic transition in AD initiation and progression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Among the infiltrating inflammatory cells, macrophages are pivotal. Under pathological stimuli such as hypertension, they become activated and often polarize toward the pro-inflammatory M1 phenotype [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These macrophages further aggravate aortic injury and vascular remodeling through the secretion of inflammatory mediators and matrix metalloproteinases (MMPs), which compromise the structural integrity of the extracellular matrix (ECM) and accelerate AD progression [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, inflammatory factors or mechanical stress can prompt VSMCs to switch from contractile to synthetic phenotypes [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This phenotypic transition reduces their contractile function while increasing proliferation, migration, and synthetic activities, collectively leading to deterioration of the aortic wall [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Notably, intercellular communication between macrophages and VSMCs is vital in AD pathogenesis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Although previous studies suggested that M1 macrophages affect VSMC phenotype switching and vascular integrity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the precise molecular mechanisms of this interaction remain unclear.\u003c/p\u003e \u003cp\u003eEmerging evidence indicates that exosomes function as essential vehicles for orchestrating cross-talk among cells during cardiovascular disorders by transferring bioactive cargos, including miRNAs, over long distances [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. While macrophage-derived exosomes are known to participate in the pathogenesis of vessel-related disorders, including atherosclerosis and neointimal hyperplasia via miRNAs (e.g., miR-21-3p, miR-222) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], their contribution to AD progression\u0026mdash;and in particular the specific miRNA effectors regulating VSMC phenotype in vivo\u0026mdash;remains largely unexplored. Notably, Notably, how M1 macrophage-sourced exosomal miRNAs directly regulate the SMAD signaling pathway in VSMCs remains an open question.\u003c/p\u003e \u003cp\u003eIn this work, we provide the first evidence that that M1 macrophage-derived exosomes deliver miR-155-5p to VSMCs, where it targets and suppresses SMAD5, thereby promoting synthetic phenotypic switching and exacerbating AD progression. Furthermore, we developed an engineered exosome-based delivery system carrying Antago-miR-155-5p, which effectively mitigates disease severity in a BAPN-induced mouse model. These findings uncover a novel macrophage-VSMC communication axis in AD pathogenesis and provide a promising foundation for exosome-mediated RNA interference as a precise clinical intervention.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eCell culture and transfection reagents: The essential media and supplements for cell maintenance, including high-glucose Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin-EDTA, and 100\u0026times; penicillin-streptomycin, were procured from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). For transfection purposes, Lipofectamine 3000 and Opti-MEM\u0026trade; I Reduced-Serum Medium were acquired through Invitrogen and Gibco (Thermo Fisher Scientific). The miR-155-5p mimics (including FAM-tagged versions), Antago-miR-155-5p, and respective negative control sequences (NC mimics and Antago-NC) were custom-synthesized by RiboBio Co., Ltd. (Guangzhou, China). The SMAD5-expressing plasmid was provided by Tsingke Biotechnology Co., Ltd.\u003c/p\u003e \u003cp\u003eMacrophage polarization and stimulation reagents: Lipopolysaccharide (LPS, catalog no. BS904) was purchased from Biosharp (Shanghai, China). Recombinant mouse interferon-gamma (IFN-γ, catalog no. HY-P70610) was from MedChemExpress (Monmouth Junction, NJ, USA). Angiotensin II (Ang II; cat. no. A9525) was sourced through Sigma-Aldrich (Shanghai, China).Regarding exosome-related materials, the secretion inhibitor GW4869 was acquired from MedChemExpress, while the PKH26 Red Fluorescent Cell Linker Kit was provided by Yu Jiubo Co., Ltd. (Shanghai, China).\u003c/p\u003e \u003cp\u003eAntibodies: Specific primary antibodies targeting CD9, CD63, TSG101, Calponin (CNN), and inducible nitric oxide synthase (iNOS) were purchased via Abcam (Cambridge, UK). Antibodies against SM22α, CD68, F4/80, α-SMA, and CD206 were from Proteintech Group, Inc. (Wuhan, China). The anti-MYOCD antibody was sourced from Sigma-Aldrich; furthermore, ABclonal Technology Co., Ltd. (Wuhan, China) provided the antibodies for SMAD5, E2F2, and SATB1.\u003c/p\u003e \u003cp\u003eOther reagents:Protease and phosphatase inhibitor cocktails were from Sigma-Aldrich.\u003c/p\u003e \u003cp\u003eTRIzol reagent was from Invitrogen.β-aminopropionitrile monofumarate (BAPN, catalog no. S42995) was from Yuanye Bio-Technology (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Human tissue study\u003c/h2\u003e \u003cp\u003eSpecimens of aortic tissue, comprising both sporadic aortic dissection cases and healthy controls, were retrieved from our institutional biobank. We excluded individuals presenting with hereditary aortic conditions (e.g., Marfan syndrome) or dissections involving the mitral or aortic valves to maintain cohort homogeneity. For the control group, aortic tissues were harvested during cardiac transplantations from age-matched donors exhibiting no history of aortic coarctation, aneurysm, dissection, or prior surgical intervention. This study adhered to the Declaration of Helsinki ethical standards, with all protocols involving human material receiving formal endorsement from the Human Research Ethics Committee at Zhongnan Hospital, Wuhan University(Approval No. 2023187).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Animal study\u003c/h2\u003e \u003cp\u003e All animal-related experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals. The study design underwent ethical review and approval by the Animal Care and Use Committee of Zhongnan Hospital, Wuhan University (No. ZN2023009). The animals were maintained under a standard light/dark cycle, with ad libitum access to standardized chow and water. To establish the AD model, three-week-old male mice received 1 g/kg/day of BAPN [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] (Yuanye Bio-Technology, Shanghai, China) via their drinking supply over a 4-week duration.\u003c/p\u003e \u003cp\u003eMice were allocated into experimental cohorts (n\u0026thinsp;=\u0026thinsp;15 per group) through a randomization process. Treatment delivery was performed through either intraperitoneal (i.p.) injection or intravenous (i.v.) infusion every 48 hours from week 3 to week 7 (end of experiment).GW4869 intervention Groups: Saline\u0026thinsp;+\u0026thinsp;DMSO, Saline\u0026thinsp;+\u0026thinsp;GW4869, BAPN\u0026thinsp;+\u0026thinsp;DMSO, BAPN\u0026thinsp;+\u0026thinsp;GW4869. GW4869 (20 \u0026micro;M stock, diluted appropriately) or vehicle (DMSO) was given i.p.Exosome administration Groups: Saline\u0026thinsp;+\u0026thinsp;M0-Exos, Saline\u0026thinsp;+\u0026thinsp;M1-Exos, BAPN\u0026thinsp;+\u0026thinsp;M0-Exos, BAPN\u0026thinsp;+\u0026thinsp;M1-Exos.Purified M0-Exos or M1-Exos (20 \u0026micro;g protein in 150 \u0026micro;L PBS) were injected via tail vein.Antago-miR-155-5p exosome therapy Groups: BAPN\u0026thinsp;+\u0026thinsp;Antago-miR-NC-Exos, BAPN\u0026thinsp;+\u0026thinsp;Antago-miR-155-5p-Exos.Engineered exosomes (M0-derived, loaded with Antago-miR-155-5p or negative control, 20\u0026micro;g protein/150 \u0026micro;L) were administered i.v. via tail vein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Isolation and culture of primary mouse VSMCs\u003c/h2\u003e \u003cp\u003eUnder sterile conditions, thoracic aortic tissues were harvested from male C57BL/6 mice at 10 weeks of age[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. After euthanasia (cervical dislocation) and 70% ethanol immersion, the aorta was perfused with PBS via left ventricular puncture, dissected, and adventitia/fat removed under a stereomicroscope. Cleaned aortas were cut into 1\u0026ndash;2 mm\u0026sup2; pieces in serum-free high-glucose DMEM, following a 60-minute enzymatic digestion (0.25% trypsin and collagenase) maintained at 37\u0026deg;C, the reaction was neutralized using 10% FBS-supplemented DMEM. After centrifugation, the resulting cell pellet was homogenized in complete growth medium and subsequently distributed into six-well plates pre-coated with 0.1% gelatin.The primary cultures were maintained in a humidified 5% CO2 environment at 37\u0026deg;C. Initial media replacement occurred after a 3-day quiescent period, followed by regular changes every 72 hours. Experimental assays utilized cells between the second and third passages.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell culture and transfection\u003c/h2\u003e \u003cp\u003eThe RAW264.7 cells were procured from the Cell Bank in Shanghai, China, which is part of the Chinese Academy of Sciences. Mice at eight weeks of age had their thoracic aortas used to generate VSMCs. We cultured RAW264.7 cells and primary VSMCs in high-glucose DMEM with 10% FBS and 1% P/S. Additional analysis was conducted using VSMCs collected at passages two to three. Cells were serum-starved overnight in DMEM with 0.1% FBS, then treated with 1 \u0026micro;M Angiotensin II (Ang II, Sigma, #A9525) for 24 hours prior to experiments. To distribute miR-155-5p mimics (50 nM), miR-155-5p inhibitors (100 nM), or SMAD5 plasmids (2 \u0026micro;g/mL), transfection was carried out using Lipofectamine 3000 (Invitrogen, #L3000015) at a cell density of about 60%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Induction of M0 and M1 macrophages\u003c/h2\u003e \u003cp\u003eRAW264.7 macrophages were maintained in high-glucose DMEM containing 1% penicillin-streptomycin and 10% FBS. Upon achieving a confluency of nearly 60%, the growth medium was replaced by DMEM supplemented with 10% exosome-depleted FBS. To induce M1 polarization, macrophages were stimulated for a 24-hour period using 20 ng/mL IFN-γ (MedChemExpress, #HY-P70610) and 100 ng/mL LPS (Biosharp, #BS904). Collecting macrophages for phenotypic evaluation and observing cell morphology following activation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Exosome purification\u003c/h2\u003e \u003cp\u003eFollowing recognized protocols, differential ultracentrifugation was employed to isolate exosomes from the collected culture supernatants. RAW264.7 macrophages were plated uniformly on 150-mm culture plates. At approximately 60% confluency, the medium was exchanged for DMEM containing 10% exosome-free FBS. After a 24-hour activation with 20 ng/mL IFN-γ and 100 ng/mL LPS, the cells underwent a medium exchange to serum-free DMEM for an additional 48-hour incubation.\u003c/p\u003e \u003cp\u003eThe harvested conditioned media was initially subjected to centrifugation at 300 \u0026times;g (10 min, 4\u0026deg;C), followed by a subsequent round at 2,000\u0026times;g (30 min, 4\u0026deg;C) to eliminate cellular debris and detached cells. After passing the supernatant through a 0.22 \u0026micro;m membrane filter (Merck Millipore), the filtrate was further purified via centrifugation at 10,000\u0026times;g for 30 minutes at 4\u0026deg;C. Final exosome recovery was achieved by ultracentrifugation (Beckman Coulter SW28 rotor) at \u003cspan\u003e$\u003c/span\u003e120,000\u0026times;g for 90 minutes at 4\u0026deg;C. The resulting pellet was then resuspended in 100 \u0026micro;L of sterile PBS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Transmission electron microscopy (TEM) imaging\u003c/h2\u003e \u003cp\u003eTo analyze the shape of the separated exosomes, TEM was used. The exosome suspension, which was 20 \u0026micro;L in volume, was spread out on copper grids and left to incubate for 5\u0026ndash;10 minutes. The grids were sponged with filter paper to soak up any extra fluid, allowed to air dry for a short while, and then negatively stained with 20 \u0026micro;L of 2% phosphotungstic acid for 5 minutes. After removing any excess staining solution, the grids were carefully dried under an incandescent bulb. The images were taken using an 80 kV accelerating voltage on a Hitachi H-7650 TEM (Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Nanoparticle tracking analysis (NTA)\u003c/h2\u003e \u003cp\u003eNTA was performed with a ZetaView PMX110 (Particle Metrix) to assess exosome size and concentration. Prior to analysis, the chamber underwent triple rinsing with purified water. In order to calibrate the measuring chamber, exosome samples were diluted in PBS and then added. After the calibration, the chamber was filled with 1\u0026ndash;2 \u0026micro;L of a diluted exosome solution in 2 mL of PBS. A 405 nm laser was used to take measurements while a 60-second video was recorded at 30 frames per second. This allowed for the ability to follow particles in real-time. Particle concentration and size distribution were analyzed by ZetaView software (version 8.02.28), utilizing Brownian motion principles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9. \u003cb\u003eWestern blot (WB) for exosomes\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eExosomes were characterized by WB using antibodies against exosomal markers CD63, CD9, and TSG101 (all Abcam, 1:1000). Standard procedures were followed for protein extraction, electrophoresis, transfer, and immunoblotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10. PKH26 labeling and exosome uptake by VSMCs\u003c/h2\u003e \u003cp\u003eIsolated exosomes were fluorescently labeled using PKH26 dye, after which they were incubated with VSMCs for 12 hours. Cellular uptake of labeled exosomes was visualized through confocal microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Non-contact co-culture of RAW264.7 and VSMCs\u003c/h2\u003e \u003cp\u003eA non-contact Transwell co-culture assay was established using 0.4 \u0026micro;m polyethylene membrane inserts (Corning). We seeded RAW264.7 macrophages into the apical compartments, whereas VSMCs occupied the basal chambers of the Transwell system. Upon achieving approximately 60% confluency. Activation of RAW264.7 cells was achieved by exposure to 20 ng/mL IFN-γ, 100 ng/mL LPS, and 20 \u0026micro;M GW4869 (MCE), whereas PBS treatment was applied as a control. Following a 24-hour interval, the existing medium was swapped for serum-depleted high-glucose DMEM, extending the incubation period by an additional 48 hours. Subsequently, VSMCs were harvested for further evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.12. miRNA sequencing and differential expression analysis\u003c/h2\u003e \u003cp\u003eWe isolated total RNA from both M0/M1-polarized macrophages and their respective exosomal fractions. Evaluation of RNA integrity and concentration was performed via an Agilent 2200 Bioanalyzer. Shanghai Huaying Biotechnology (Shanghai, China) handled library construction and subsequent miRNA profiling, utilizing the Illumina HiSeq 2500 system. Differential expression analysis of miRNAs was conducted using standard bioinformatics pipelines. Criteria for identifying significant miRNA dysregulation included an adjusted P-value below 0.05 and a minimum absolute log2-fold change of 1. To verify the levels of exosomal miR-155-5p, qRT-PCR was executed on a Bio-Rad CFX96 Real-Time PCR platform (Bio-Rad, USA) employing the Vazyme ChamQ SYBR Master Mix.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.13. miR-155-5p translocation analysis\u003c/h2\u003e \u003cp\u003eTo trace exosomal trafficking, RAW264.7 macrophages underwent an initial 48-hour transfection with FAM-labeled miR-155-5p mimics, subsequently followed by induction toward the M1 state. After recovering exosomes from the conditioned media, they were stained with PKH26 and added to VSMC cultures for a 12-hour incubation period. After fixation, cytoskeletal and nuclear staining were performed, and confocal microscopy was employed to visualize intracellular localization of FAM-miR-155-5p. In associated experiments, exosomes originating from M1 macrophages (M1-Exos) were permeabilized using 0.5% Triton X-100 for 5 minutes, succeeded by RNA degradation with RNase (20 U/\u0026micro;L) for one hour. After PBS rinses, the exosomes were subjected to ultracentrifugation at 120,000 \u0026times; g for 90 minutes (4\u0026deg;C) to eliminate residual RNase and Triton X-100. Subsequently, miR-155-5p levels in recipient VSMCs were measured through qPCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Bioinformatics analysis\u003c/h2\u003e \u003cp\u003eRNA sequencing was performed on aortic tissues from BAPN-induced AD mice and age-matched controls by Shanghai Huaying Biotechnology Co., Ltd. (Shanghai, China). Identification of differentially expressed genes (DEGs) was performed through the limma and edgeR R toolkits. The selection criteria were defined as an adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (applying Benjamini-Hochberg correction) and a |log2 fold change| of at least 1. miR-155-5p target genes were predicted using TargetScan (v7.2) and miRTarBase (v8.0), focusing on conserved 3\u0026rsquo;UTR binding sites. Candidate targets were obtained by intersecting the predicted targets with significantly downregulated DEGs from the RNA-seq dataset. To explore the biological roles of these overlapping gene candidates, we performed functional enrichment analysis via the Metascape platform, utilizing standard settings, including GO, KEGG, and Reactome databases. Statistical significance was assigned to enriched functional terms exhibiting an adjusted P-value below 0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.15. Luciferase reporter assay\u003c/h2\u003e \u003cp\u003eTo construct the reporter vectors, either the wild-type (WT) 3\u0026rsquo;UTR of SMAD5, which harbors the forecast miR-155-5p binding site, or its mutant (Mut) counterpart with a scrambled seed sequence, was inserted into the pGL6-basic luciferase plasmid (Promega) at a position following the firefly luciferase gene. All constructs were verified by Sanger sequencing. Primary VSMCs underwent plating in 24-well clusters and were subsequently co-transfected upon reaching 60\u0026ndash;70% density. The transfection mixture comprised 200 ng of WT/Mut reporter plasmids, a 50 nM concentration of miR-155-5p mimics (or NC mimics), and 20 ng of pRL-TK (Promega) serving as the endogenous reference, facilitated by Lipofectamine 3000 (Invitrogen) according to the manufacturer\u0026rsquo;s instructions. Forty-eight hours post-transfection, cell lysis was performed to facilitate the sequential determination of firefly and Renilla luciferase signal intensities through the Dual-Luciferase Reporter Assay System (Promega) via a GloMax luminometer (Promega). Normalization of firefly luciferase levels against Renilla luciferase was conducted... All assays were executed in triplicate and replicated a minimum of three independent times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e2.16. Immunofluorescence (IF) assay\u003c/h2\u003e \u003cp\u003eTissue immunofluorescence: Aortic samples were initially preserved in 4% paraformaldehyde (PFA) for a 24-hour period, followed by embedding in paraffin and preparation of 5-\u0026micro;m thick slices. These sections then underwent deparaffinization using xylene, rehydration via descending ethanol concentrations, and heat-induced epitope retrieval within a pH 6.0 citrate buffer environment using microwave heating for 10\u0026ndash;15 min. After blocking with 10% bovine serum albumin (BSA) diluted in PBS for 1 hour at ambient temperature. This was followed by an overnight incubation at 4\u0026deg;C with specific primary antibodies... After three PBST washing cycles, the specimens were exposed to corresponding fluorescent secondary antibodies for a duration of 2 hours at room temperature. For nuclear visualization, DAPI (1 \u0026micro;g/mL) was applied for 5 minutes. Slides were secured using an antifade mounting medium prior to visualization under either a Leica fluorescence or confocal microscope. We utilized ImageJ software to quantify the colocalization and fluorescence intensities.\u003c/p\u003e \u003cp\u003eCellular immunofluorescence: Primary VSMCs or treated cells grown on coverslips underwent fixation in 4% paraformaldehyde for a duration of 15 min at ambient temperature, followed by a 10-minute permeabilization step utilizing 0.2% Triton X-100 (diluted in PBS). Subsequent to a 1-hour blocking period in 10% BSA, the specimens were subjected to overnight incubation with primary antibodies at 4\u0026deg;C. Exposure to secondary fluorescent antibodies was performed for 2 h under room temperature conditions. A Leica confocal microscope was employed for image acquisition, with subsequent quantification conducted via ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.17. RNA extraction and quantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from the samples utilizing the TRIzol reagent (purchased from Invitrogen). A NanoDrop One spectrophotometer was utilized to evaluate both the quantity and optical purity of the extracted RNA, which is made by Thermo Fisher Scientific. The HiScript III RT SuperMix (Vazyme) and 1 \u0026micro;g reverse transcription was executed starting with a 1 \u0026micro;g template of total RNA. Quantitative PCR assays were carried out on a Bio-Rad CFX96 Real-Time PCR detection system, employing the ChamQ SYBR Master Mix (Vazyme). GAPDH served as the internal control for normalizing gene expression, which was subsequently determined via the \u003cspan\u003e$\u003c/span\u003e2^{-\\Delta\\Delta CT}\u003cspan\u003e$\u003c/span\u003e analytical approach.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e2.18. WB\u003c/h2\u003e \u003cp\u003eProtein extraction was achieved by lysis in RIPA buffer supplemented with a cocktail of phosphatase and protease inhibitors from cultured cells and aortic tissue samples. Protein concentrations were determined by employing the BCA protein assay kit. Proteins of the same amount were electrophoretically separated on SDS-PAGE gels before being deposited onto PVDF membranes. Following a 1-hour blocking step in 5% non-fat milk at ambient temperature, the blots were exposed to specific primary antibodies overnight while maintained at 4\u0026deg;C. Membranes were subsequently incubated with HRP-conjugated secondary antibodies for 1 hour following multiple rinse cycles in TBST buffer. The protein signals were detected through ECL chemiluminescence and the resulting band intensities were processed using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e2.19. Statistical analysis\u003c/h2\u003e \u003cp\u003eData processing and statistical assessments were executed utilizing GraphPad Prism (v10.0). Results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD), except where specifically noted. The Shapiro-Wilk test was employed to verify whether the data followed a normal distribution. Comparisons utilized independent Student\u0026rsquo;s t-tests, or one-way ANOVA coupled with Tukey\u0026rsquo;s multiple comparison tests. Correlation assessments were performed by calculating Pearson\u0026rsquo;s correlation coefficients. Differences were considered statistically significant when the p-value was below 0.05. Blinding and randomization strategies were systematically employed during experiments and result evaluations. *: P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **: P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***: P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and ****: P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001denote varying levels of statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Accumulation of M1-polarized Macrophages and Exosomes in Aortic Dissection (AD) Tissue\u003c/h2\u003e \u003cp\u003eAlthough exosomes are known mediators of intercellular communication in cardiovascular disease[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], their role in AD remains poorly understood. Therefore, IF staining was performed to analyze macrophage and exosome distribution in human and mouse AD tissues compared with normal controls. In human tissues, AD samples showed marked infiltration of macrophages (CD68+, red) and elevated expression of the exosomal marker TSG101 (green), with strong colocalization in merged images (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Quantification revealed significantly higher relative fluorescence intensity for both TSG101 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) and CD68 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) in AD tissues compared to controls. Similar findings were observed in mouse aortic tissues, where AD samples exhibited increased TSG101 (green) and macrophage marker F4/80 (red) expression, with evident colocalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Quantitative analysis confirmed significantly elevated fluorescence intensity for TSG101 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE) and F4/80 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) in AD versus control tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther, macrophage polarization was further assessed using markers for pro-inflammatory M1 (iNOS, red) and anti-inflammatory M2 (CD206, green) phenotypes. In human AD tissues, iNOS expression was substantially increased, while CD206 was decreased, indicating a shift toward M1 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Quantification showed significantly higher iNOS intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and lower CD206 intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI) in AD tissues compared to controls.In mouse AD tissues, a similar M1-dominant polarization was evident, with elevated iNOS and reduced CD206 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Relative fluorescence intensity was significantly increased for iNOS (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK) and decreased for CD206 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL) in AD versus control tissues. These results indicate a critical role of M1 macrophages in AD pathogenesis. These findings highlight the pivotal contribution of M1 macrophages to AD development, suggesting a substantial accumulation of both M1 cells and exosomes in AD tissues from both humans and mice, with close spatial association between macrophages and exosomes. This suggests that M1 macrophages may contribute to AD progression through exosome-mediated intercellular communication.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Inhibition of exosome secretion by GW4869 attenuates BAPN-induced aortic dissection progression in mice\u003c/h2\u003e \u003cp\u003eA mouse model of AD was established by administering β-Aminopropionitrile (BAPN) to induce disease, followed by treatment with the exosome secretion inhibitor GW486932[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] or DMSO (vehicle) every 48 hours via intraperitoneal injection for 4 weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). BAPN administration significantly increased mortality compared to saline controls, with survival curves showing a marked decline in the BAPN+DMSO group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Treatment with GW4869 significantly improved survival in BAPN-treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Consistent with the survival data, mortality rates were 66.7% in the BAPN+DMSO group and reduced to 33.3% in the BAPN+GW4869 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGross morphological examination at week 7 revealed severe aortic dilation, tortuosity, and frequent intramural hematoma/rupture in BAPN+DMSO-treated mice, whereas GW4869 treatment substantially preserved aortic architecture and reduced macroscopic lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Quantification confirmed a significantly lower AD incidence in the BAPN+GW4869 group (46.7%) compared to BAPN+DMSO (86.7%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Maximum aortic diameter was markedly increased in BAPN+DMSO mice but significantly attenuated by GW4869 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Histological analyses further confirmed that GW4869 alleviated structural changes caused by BAPN, including luminal expansion (H\u0026amp;E staining), elastic fiber fragmentation (EVG staining), and collagen deposition (Masson staining) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and H).\u003c/p\u003e \u003cp\u003eThese results indicate that inhibition of exosome secretion with GW4869 significantly mitigates BAPN-induced AD progression, including reduced mortality, AD incidence, aortic dilation, elastic fiber degradation, and structural remodeling. This suggests that macrophage-derived exosomes play a critical pathogenic role in AD development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Characterization and purification of extracellular vesicles from macrophages\u003c/h2\u003e \u003cp\u003eTo investigate the role of macrophage-derived exosomes in AD, M1 polarization of RAW264.7 cells was induced using 100 ng/mL LPS and 20 ng/mL IFN-γ. Purification of exosomes from conditioned medium of M0 (M0-Exos) and M1 (M1-Exos) macrophages was performed after polarization. At the optimal concentrations of 100 ng/mL LPS and 20 ng/mL IFN-γ, the expression of iNOS, a marker specific to M1, was significantly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and C). Additionally, the macrophages underwent noticeable morphological alterations, with multipolar extensions, in contrast to the smaller and spherical control macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). WB confirmed successful M1 polarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and D). Exosomes were isolated via ultracentrifugation, displaying high expression of exosomal markers (TSG101, CD9, CD63) without contamination by endoplasmic reticulum(ER) marker Calnexin or cytoplasmic marker GAPDH (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Transmission electron microscopy showed exosomes had typical bilayer membranes and a mean diameter centering around 100 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Nanoparticle tracking analysis corroborated this, with both M0-Exos and M1-Exos exhibiting size distributions peaking at approximately 100 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and I). Collectively, these characterizations validate the successful generation of M1-polarized macrophages and isolation of high-purity exosomes, setting the foundation for evaluating their functional impact on AD progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Exosomes derived from M1 macrophages promote phenotypic transition of vascular smooth muscle cells (VSMCs)\u003c/h2\u003e \u003cp\u003eIn the pathogenesis of AD, intercellular crosstalk between macrophages and VSMCs plays a pivotal role, with exosomes potentially serving as key mediators, and VSMC phenotypic switching from contractile to synthetic states being essential for disease initiation and progression[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. To determine whether M1 macrophage-derived exosomes influence phenotypic switching in VSMCs, primary mouse aortic VSMCs were exposed to exosomes isolated from M0- or M1-polarized RAW264.7 cells. Initially, primary VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) were incubated with PKH26-labeled M1-Exos or control M0-Exos for 24 hours. Cellular uptake of PKH26-labeled exosomes (red) by VSMCs was visualized using confocal microscopy, with VSMCs labeled for F-actin (green) and nuclei (blue) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Protein-level assessment by Western blotting revealed diminished expression of contractile markers (MYOCD, α-SMA, CNN, SM22α) in M1-Exos-treated VSMCs compared to M0-Exos (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), with densitometric quantification confirming statistically significant decreases (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Transcriptional profiling via qRT-PCR similarly showed suppressed mRNA levels for MYOCD, SM22α, α-SMA, and CNN in the M1-Exos group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026ndash;H)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo verify exosome-specific effects, a non-contact co-culture system using RAW264.7 macrophages (upper chamber) and VSMCs (lower chamber) was established (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). LPS/IFN-γ-polarized M1 macrophages markedly reduced the expression of VSMCs contractile markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-N). However, addition of the exosome secretion inhibitor GW4869 (20 \u0026micro;M) partially restored contractile marker expression without altering M0 baselines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ-N). Together, this highlights a critical mechanism whereby M1-Exos facilitate VSMC transdifferentiation. This exosome-mediated crosstalk is pivotal in AD pathogenesis, implicating it as a potential therapeutic target.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e3.5. M1-derived exosomes exacerbate BAPN-induced AD progression and promote phenotypic switching in VSMCs\u003c/h2\u003e \u003cp\u003eTo further confirm the functional roles of macrophage-derived exosomes in AD, exosomes secreted by RAW264.7 macrophages polarized into either the M1 (M1-Exos) or M0 (M0-Exos) states were isolated and characterized. An AD model was subsequently induced in mice via BAPN administration, followed by intravenous injection of either M1-Exos or control M0-Exos (20\u0026micro;g protein/150 \u0026micro;l) every 48h starting at week 3 until termination at week 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). M1-Exos treatment significantly increased mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), accelerated aortic rupture (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and intensified aortic dilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and F) compared with the M0-Exos group. Morphometric analysis confirmed an increased maximum aortic diameter in mice treated with M1-Exos (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and F) and higher incidence of AD (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Tissue-level examination via H\u0026amp;E staining uncovered extensive cavity enlargement and wall thinning in BAPN+M1-Exos sections, while EVG staining exposed severe elastic lamina disruption (arrows marking fractures) and Masson's trichrome indicated heightened fibrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Molecular profiling of aortic extracts by Western blotting showed reduced levels of contractile indicators (MYOCD, α-SMA, CNN, SM22α) in BAPN+M1-Exos tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI), with densitometry confirming significant declines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ). These results indicate that M1-Exos intensify AD severity in vivo, amplifying structural damage and VSMC dedifferentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e3.6. M1-derived exosomes induce phenotypic switching of VSMCs through miR-155-5p delivery\u003c/h2\u003e \u003cp\u003eExosomes mediate intercellular communication by transporting bioactive cargo such as miRNAs and proteins, influencing cellular signaling pathways and functions, thereby contributing to disease pathogenesis[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Given previous findings that M1-Exos exacerbate AD, we sought to identify the key functional exosomal components responsible for this effect. we conducted miRNA sequencing on exosomes from M1- and M0-polarized RAW264.7 macrophages, complemented by analysis of public datasets. Heatmap visualization based on sequencing data of exosomes derived from M0 and M1 macrophages showed the upregulation of specific miRNAs in M1 cells, including a prominent increase in miR-155-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Volcano plots from our exosomal sequencing highlighted differentially expressed miRNAs, with miR-155-5p markedly upregulated in M1-Exos (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). Integration with GSE125171 (BMDMs) revealed consistent patterns: heatmaps and volcano plots showed miR-155-5p among top upregulated miRNAs in M1-BMDMs (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB and C). Our cellular sequencing of RAW264.7 similarly identified miR-155-5p enrichment in M1 states via heatmaps and volcano plots (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD and E). A Venn diagram cross-referencing predicted targets, GSE125171 differentials, and our exosomal miRNAs pinpointed miR-155-5p as the overlapping hit (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Therefore, we hypothesized that M1-Exos accelerate AD by transferring miR-155-5p to VSMCs and inducing a synthetic phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo test this hypothesis, miR-155-5p expression was first verified by qRT-PCR, which showed significant upregulation in M1 macrophages stimulated by LPS/IFN-γ and their exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and D). Elevated miR-155-5p expression was confirmed in human and murine AD tissues compared to normal controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and F). RNA-FISH further revealed notable accumulation of miR-155-5p in AD tissues, consistent with sequencing data (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and H).\u003c/p\u003e \u003cp\u003eNext, M1 macrophages were transfected with FAM-labeled miR-155-5p mimics, and exosomes isolated from conditioned media were incubated with VSMCs. Delivery experiments confirmed exosomal shuttling: FAM-miR-155-5p (green) overlapped with PKH26-Exos (red) in VSMC cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Additionally, qRT-PCR confirmed elevated miR-155-5p levels within treated VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Pretreatment of exosomes with Triton X-100, which disrupts membranes prior to RNase digestion, significantly reduced miR-155-5p levels transferred into VSMCs. However, RNase treatment alone had little effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK). These results suggest that exosomal packaging protects miR-155-5p from degradation, facilitating functional delivery.\u003c/p\u003e \u003cp\u003eExosomes (miR-155\u003csup\u003eOE\u003c/sup\u003e-Exos) were extracted from RAW264.7 macrophages that had been engineered to stably express high levels of miR-155-5p (miR-155\u003csup\u003eOE\u003c/sup\u003e), in order to evaluate the functional significance of miR-155-5p. The results of the qRT-PCR assay confirmed the increased expression of miR-155-5p in both cells and exosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL and M). WB examination (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN and R) indicated that incubating VSMCs with miR-155\u003csup\u003eOE\u003c/sup\u003e-Exos significantly reduced levels of contractile phenotypic markers such as α-SMA, SM22α, CNN, and MYOCD, which is similar to the effect seen with exosomes produced from M1. These findings demonstrate that M1-derived exosomes promote VSMC phenotype switching through miR-155-5p transfer, contributing to AD progression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e3.7. miR-155-5p negatively regulates SMAD5 expression to control VSMC phenotypic switching\u003c/h2\u003e \u003cp\u003eTo uncover the downstream mechanism by which exosomal miR-155-5p induces VSMC dedifferentiation, we integrated RNA-seq data from treated VSMCs with miRNA target prediction databases (miRDB and TargetScan). Venn analysis revealed 17 overlapping differentially expressed mRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Network mapping prioritized SMAD5 as a high-confidence target with strong connectivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Enrichment analysis showed these candidates were involved in transcriptional regulation, protein phosphorylation, angiogenesis, and morphological transformation, relevant to AD pathogenesis (Fig.S2A-C). GO enrichment analysis of DEGs in miR-155\u003csup\u003eOE\u003c/sup\u003e-Exos-treated VSMCs revealed significant overrepresentation of terms related to transcription factor activity, DNA-binding regulation, and cell homeostasis (Fig. S3A), further supporting a role in transcriptional reprogramming during phenotypic switching.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next screened 17 candidate target genes in primary VSMCs exposed to miR-155\u003csup\u003eOE\u003c/sup\u003e-Exos versus miR-155\u003csup\u003eNC\u003c/sup\u003e-Exos using qRT-PCR. Among them, E2F2, SMAD5, STAB1, PICLAM, KDM3A and SDCBP showed consistent and significant downregulation (Fig. S3N-S), whereas other candidates (HIF1A, ITK, TAB2, GNAS, etc.) exhibited no significant or inconsistent changes (Fig. S3C\u0026ndash;M). Tissue-level validation confirmed these findings: qRT-PCR screening of candidate genes (E2F2, SMAD5, STAB1, PICLAM, KDM3A, SDCBP) in both human and mouse AD aortic tissues (Fig. S4A). In human samples, SMAD5, STAB1, E2F2 and PICLAM mRNA levels were significantly reduced in AD compared to controls (Fig. S4B\u0026ndash;e), whereas KDM3A and SDCBP showed no consistent change (Fig. S4F and G). Similar downregulation of SMAD5, STAB1, and E2F2 was observed in mouse AD tissues (Fig. S4H\u0026ndash;J), with KDM3A, PICLAM and SDCBP unchanged (Fig. S4L\u0026ndash;M). WB screening of candidate genes (E2F2, SMAD5, STAB1) in both human and mouse AD aortic tissues (Fig. S5A). In mouse samples, SMAD5 and STAB1 protein levels were significantly reduced in AD compared to controls (Fig. S5B-D), whereas E2F2 showed no consistent change (Fig. S5E). Similar downregulation of SMAD5 was observed in human AD tissues (Fig. S5F and G), with E2F2 and STAB1 unchanged (Fig. S5H and I). Validation pipeline combined qRT-PCR for mRNA candidates in treated VSMCs and AD tissues, followed by protein confirmation via WB in human and mouse AD samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Among candidates, SMAD5 showed consistent downregulation. IF staining further corroborated reduced SMAD5 and SM22α expression in AD aortic walls from humans and mice, with diminished colocalization in merged images (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and E). Quantitative fluorescence analysis confirmed significant downregulation of both proteins in AD tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-I).\u003c/p\u003e \u003cp\u003eTargetScan bioinformatic predictions and dual-luciferase assays provided evidence confirming direct interaction of miR-155-5p with the SMAD5 3\u0026prime;-UTR region (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ, K). To further establish the functional consequence of this interaction in the context of exosomal delivery, primary VSMCs were treated with exosomes derived from miR-155-overexpressing RAW264.7 cells (miR-155\u003csup\u003eOE\u003c/sup\u003e-Exos). Immunoblot analysis further demonstrated reduced SMAD5 protein levels upon miR-155-5p upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL and M). Rescue experiments indicated SMAD5 overexpression in miR-155-5p-treated VSMCs restored expression of contractile markers (α-SMA, SM22α, CNN, MYOCD) suppressed by miR-155-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN\u0026ndash;S). Collectively, these results demonstrate that exosomal miR-155-5p targets SMAD5 to induce synthetic phenotypic switching in VSMCs, thereby contributing to AD progression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Engineered Antago-miR-155-5p exosomes suppress AD progression by restoring SMAD5 and contractile phenotype\u003c/h2\u003e \u003cp\u003eOur results demonstrated that exosomal miR-155-5p from M1 macrophages promoted BAPN-induced AD through VSMC phenotypic switching by targeting SMAD5. Given previous reports of elevated serum miR-155-5p in AD patients, suggesting its potential as a diagnostic biomarker [55], we hypothesized that exosomal miR-155-5p promotes AD via regulation of SMAD5 and VSMC phenotype.\u003c/p\u003e \u003cp\u003eTo evaluate these findings in vivo, mice were subjected to BAPN-induced AD modeling. Animals were randomized into two experimental cohorts (n\u0026thinsp;=\u0026thinsp;15/group) receiving intravenous administration of exosomes loaded either with Antago-miR-155-5p (Antago-miR-155-5p-Exos) or corresponding negative controls (Antago-miR-NC-Exos) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Antago-miR-155-5p-Exos significantly reduced mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), delayed aortic rupture (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), and decreased aortic dilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, F). Anatomical and histological assessments showed reduced AD incidence (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) and mitigated BAPN-induced structural damage, including luminal dilation, elastin fragmentation, and collagen deposition (H\u0026amp;E, EVG, Masson staining) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG, H). At the molecular level,WB analysis indicated that Antago-miR-155-5p-Exos reversed reductions in contractile markers (MYOCD, α-SMA, SM22α, CNN) and restored SMAD5 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI, J). IF confirmed increased SMAD5 (green) and SM22α (red) fluorescence intensity and stronger colocalization in the media of Antago-miR-155-5p-Exos-treated aortas (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK). Quantitative analysis of fluorescence intensity verified significant restoration of both proteins (Fig. S6A and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings indicate that inhibiting exosomal miR-155-5p ameliorates BAPN-induced AD progression by restoring SMAD5 expression, maintaining the VSMC contractile phenotype, and attenuating vascular remodeling and structural damage. SMAD5 thus emerges as a critical downstream effector of miR-155-5p in AD pathogenesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we identified that M1 macrophage-derived exosomal miR-155-5p exacerbates aortic dissection (AD) progression by targeting SMAD5, thereby promoting vascular smooth muscle cell (VSMC) phenotypic switching from a contractile to a synthetic state. Notably, engineered exosomes loaded with Antago-miR-155-5p effectively reversed this pathological process, significantly reducing AD incidence, mortality, and vascular remodeling in the BAPN-induced mouse model. These findings uncover a novel macrophage-VSMC communication axis in AD pathogenesis and provide strong preclinical evidence supporting exosome-based miRNA-targeted therapy as a promising strategy for aortic dissection.\u003c/p\u003e \u003cp\u003eA growing body of evidence underscores inflammation and infiltration of immune cells as critical mechanisms in the progression of AD[\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Early-stage activation and recruitment of immune cells into the aortic wall are crucial initiating steps in AD pathogenesis[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. According to Xue et al., macrophages and monocytes serve as initial responders, facilitating subsequent infiltration by neutrophils and T lymphocytes into the vascular wall[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Macrophage accumulation strongly correlates with vascular damage and rupture38. However, AD involves complex interactions among diverse cell populations. Traditionally, macrophage\u0026ndash;VSMC communication was attributed primarily to cytokines[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Recent research identifies exosomes as essential mediators of cell-to-cell communication through delivery of bioactive molecules[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Our study further demonstrates that M1 macrophages regulate VSMC phenotype transition through exosome-mediated transfer of miR-155-5p. Exosomes, as natural carriers, enable precise intercellular regulation and show increasing promise in disease mechanisms and therapeutics[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Using multi-omics approaches and functional validation, miR-155-5p emerged as a central exosomal effector driving VSMCs toward a synthetic phenotype via suppression of SMAD5, consequently promoting AD.\u003c/p\u003e \u003cp\u003eSMAD5, a key mediator of the BMP/TGF-β signaling axis, is essential for maintaining VSMC contractile phenotype and vascular homeostasis[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our demonstration that miR-155-5p directly suppresses SMAD5 expression provides a molecular link between inflammatory exosomal signaling and VSMC dedifferentiation in AD, revealing a novel upstream regulatory node that may be amenable to therapeutic targeting.\u003c/p\u003e \u003cp\u003eLeveraging the natural biocompatibility, low immunogenicity, and homing potential of macrophage-derived exosomes[\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], we engineered an Antago-miR-155-5p delivery system that significantly ameliorated AD progression in vivo. This approach offers advantages over synthetic nanoparticles, including enhanced stability of RNA cargo and potential for endogenous cell-specific targeting, positioning exosome-mediated miRNA inhibition as a viable strategy for future AD therapeutics.\u003c/p\u003e \u003cp\u003eHowever, several limitations exist. First, the in vitro M1 polarization protocol (LPS/IFN-γ) may not fully recapitulate the heterogeneous inflammatory milieu in vivo. Second, while Antago-miR-155-5p-loaded exosomes showed robust efficacy, their long-term safety, biodistribution, and potential immunogenicity require further evaluation in larger preclinical models. Future studies using cell-specific miR-155 knockout mice and conditional SMAD5 ablation will be essential to delineate contribution and specificity.\u003c/p\u003e \u003cp\u003eIn conclusion, our study identifies and characterizes the M1 macrophage-exosome-miR-155-5p-SMAD5 axis as a critical regulator of VSMC phenotypic switching in AD. These findings improve the mechanistic understanding of AD and provide new therapeutic targets. Furthermore, this research strongly supports the clinical development of exosome-based gene therapy approaches\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, we identified that M1 macrophage-derived exosomal miR-155-5p exacerbates AD progression by targeting SMAD5, thereby promoting VSMC phenotypic switching from a contractile to a synthetic state. Notably, engineered exosomes loaded with Antago-miR-155-5p effectively reversed this pathological process, significantly reducing AD incidence, mortality, and vascular remodeling in the BAPN-induced mouse model. These findings uncover a novel macrophage-VSMC communication axis in AD pathogenesis and provide strong preclinical evidence supporting exosome-based miRNA-targeted therapy as a promising strategy for aortic dissection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupporting Information is available from the Wiley Online Library or from the author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDataset supporting the current research results can be obtained from the corresponding author on justifiable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDengwei Cao and Xinyi Li performed all experiments, data analysis and drafted the manuscript. Shaoping Zhu, Jianfeng Chen and Haoxiang Li assisted with \u003cem\u003ein vitro\u003c/em\u003e experiment. Jiajun Shi, Xiaoqi Xiong, and Yumou Wang assisted with \u003cem\u003ein vivo\u003c/em\u003e experiment. Zhe Dong assisted with human aortic tissue samples collection and reviewed the manuscript. Jinping Liu, Xinyi Li and Zhe Dong provided financial support, research design, manuscript revision, and final manuscript approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinancial support for this study was provided by the National Natural Science Foundation of China (No.82470421 to LJP; No.82472204 to LXY), Funding was also received from the Central Universities\u0026rsquo; Fundamental Research Funds (2042024kf0025\u0026nbsp;to LJP),the Hubei Provincial Key Research and Development Project (2023BCB002 to LJP),\u0026nbsp;additional grants were provided by the Hubei Provincial Natural Science Foundation (No.2022CFC026 to DZ), and the Hubei Province Health Commission(No.WJ2023M061 to DZ\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon legitimate inquiry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsolidated and clarified statements regarding both human tissue use (approved by the Ethics Committee of Zhongnan Hospital, Wuhan University, Approval No. 2023187) and animal experiments (approved by the Institutional Animal Care and Use Committee of Zhongnan Hospital, Wuhan University, Approval No. ZN2023009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eP.G. 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Oncel, Exosomes: Large-scale production, isolation, drug loading efficiency, and biodistribution and uptake, Journal of Controlled Release : Official Journal of the Controlled Release Society 347 (2022) 533-543.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aortic dissection, Exosomes, Vascular smooth muscle cell, miR-155-5p, SMAD5, Phenotypic transition","lastPublishedDoi":"10.21203/rs.3.rs-8986811/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8986811/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAortic dissection (AD) constitutes a critical and potentially lethal cardiovascular emergency, yet its underlying pathogenesis remains largely obscure. The present research aimed to elucidate the contribution of exosomes derived from M1 macrophages\u0026mdash;particularly those carrying a high load of miR-155-5p\u0026mdash;to the exacerbation of AD.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMacrophage infiltration and exosome distribution were evaluated in human and mouse AD tissues. Exosomes were isolated from M1-polarized RAW264.7 macrophages and characterized. Primary mouse VSMCs were treated in vitro, while a β-aminopropionitrile (BAPN)--established murine AD model was used for in vivo studies. Interventions included GW4869 and engineered exosomes loaded with Antago-miR-155-5p. Techniques included qRT-PCR, Western blotting, luciferase assays, RNA-FISH, and histological analyses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eM1 macrophages and their exosomes were markedly enriched in AD tissues and colocalized with macrophage markers. M1-derived exosomes were internalized by VSMCs, significantly downregulated contractile indicators (CNN, α-SMA, MYOCD, SM22α), and induced synthetic phenotypic switching via exosomal miR-155-5p targeting SMAD5 (luciferase assay). In BAPN-induced mice, inhibition of exosome secretion (GW4869) or treatment with Antago-miR-155-5p-loaded exosomes significantly reduced AD incidence (from 86.7% to 46.7%), mortality, aortic dilation, elastic fiber fragmentation, and restored contractile marker and SMAD5 expression.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eExosomal miR-155-5p originating from M1 macrophages facilitates AD development through targeting SMAD5 and driving VSMC phenotypic switching. Engineered Antago-miR-155-5p exosomes represent a novel, effective therapeutic strategy for mitigating AD, providing a foundation for exosome-based RNA interference in vascular diseases.\u003c/p\u003e","manuscriptTitle":"M1 Macrophage-Derived Exosomal miR-155-5p Exacerbates Aortic Dissection Progression via SMAD5-Mediated Regulation of Vascular Smooth Muscle Cell Phenotype","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-05 11:37:28","doi":"10.21203/rs.3.rs-8986811/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5e66f33e-44e2-469a-b56a-7dcffe4448bd","owner":[],"postedDate":"March 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-05T11:37:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-05 11:37:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8986811","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8986811","identity":"rs-8986811","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}