Transcriptome Insights into Protective Mechanisms of Ferroptosis Inhibition in Aortic Dissection

Transcriptome Insights into Protective Mechanisms of Ferroptosis Inhibition in Aortic Dissection | 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 Transcriptome Insights into Protective Mechanisms of Ferroptosis Inhibition in Aortic Dissection Chun-Che Shih, Chi-Yu Chen, Chih-Pin Chuu, Chun-Yang Huang, Chia-Jung Lu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6061217/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 Aortic dissection (AD) is a life-threatening vascular condition with limited pharmacological options. This study investigated Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, in a BAPN/Ang-II-induced mouse model of AD, revealing significant therapeutic potential. Fer-1 significantly reduced AD incidence and mortality by preserving aortic wall integrity. RNA sequencing identified 922 differentially expressed genes, with 416 upregulated and 506 downregulated. Bioinformatics analysis revealed that Fer-1 modulates key regulators, such a s MEF2C and KDM5A, impacting immune responses, oxidative stress, apoptosis, and lipid metabolism. Additionally, Fer-1 alters miRNA expression, with the upregulation of miR-361-5p and downregulation of miR-3151-5p, targeting pathways involved in inflammation, oxidative stress, and smooth muscle cell (SMC) phenotypic stability. Functional pathway analysis highlighted the inhibition of actin cytoskeleton, ILK, and IL-17 signaling, essential for SMC differentiation and extracellular matrix remodeling. Gene interaction network analysis identified 21 central molecules, including CXCR3, ACACA, and BPGM, associated with lipid metabolism, inflammation, and vascular remodeling. These findings demonstrate Fer-1's potential to mitigate ferroptosis and modulate inflammation, offering a comprehensive protective mechanism against AD and potentially other ferroptosis-driven vascular diseases. Aortic dissection Ferroptosis Inflammation Transcriptomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Aortic dissection (AD) is a life-threatening vascular condition characterized by the separation of the aortic wall layers, resulting in severe complications such as rupture and organ ischemia ( 1 ). Despite significant advancements in diagnostic imaging and surgical management, the limited understanding of AD pathogenesis hampers the development of effective early-stage intervention strategies ( 2 , 3 ). Recent studies have identified oxidative stress, inflammation, smooth muscle cell (SMC) phenotypic transition and extracellular matrix (ECM) degradation as critical contributors to AD progression ( 1 , 4 , 5 ). These pathological processes frequently culminate in cell death, further compromising the structural integrity of the aortic wall ( 6 ). Although certain agents targeting apoptosis or autophagic cell death have demonstrated protective effects in mouse models of AD, their clinical translation has yet to be achieved. These findings emphasize the pivotal role of regulated cell death in AD pathogenesis and highlight the potential of targeting these pathways as a promising therapeutic strategy ( 7 ). Among the various forms of regulated cell death, ferroptosis—an iron-dependent mechanism characterized by lipid peroxidation—has emerged as a critical factor in vascular injury and inflammation ( 7 , 8 ). Ferroptosis has been implicated in the pathogenesis of several cardiovascular diseases, including doxorubicin-induced cardiotoxicity ( 9 ), ischemia-reperfusion injury ( 10 ), heart failure ( 11 ), AD ( 7 , 12 ) and stroke ( 13 ). In AD, elevated oxidative stress creates an environment conducive to ferroptotic cell death, which may exacerbate structural damage to the aortic wall ( 8 ). However, the interplay between ferroptosis and other pathological processes in AD, such as smooth muscle cell dysfunction and ECM degradation, remains inadequately explored ( 7 , 12 , 14 ). This knowledge gap underscores the critical need for focused investigations into ferroptosis-mediated mechanisms and their potential therapeutic modulation in AD. To have better understand the therapeutic potentials of targeting ferroptosis, ferrostatin-1 (Fer-1), we have examined the effects of Fer-1, a potent ferroptosis inhibitor, in β-aminopropionitrile (BAPN) and angiotensin II (Ang-II)-induced AD mouse models. Fer-1 has demonstrated efficacy in preclinical models of oxidative stress-related diseases by scavenging lipid peroxides and preserving cellular antioxidant defenses. These mechanisms effectively mitigate oxidative damage and inflammation ( 7 , 15 , 16 ). This study aims to elucidate the molecular mechanisms underlying the therapeutic effects of Fer-1 and its role in inhibiting ferroptosis through histological, transcriptomic, and pathway analyses. Materials and methods Model establishment and interventions All animal experiments were approved by the Institutional Animal Care and Use Committee of Taipei Medical University. Male C57BL/6J mice were housed in a specific pathogen-free (SPF) facility under a 12-hour light-dark cycle and fed a standard diet. An aortic dissection (AD) model was induced in three-week-old mice by oral administration of 0.5% β-aminopropionitrile (BAPN; Sigma-Aldrich, St. Louis, MO, USA) for four weeks. Subsequently, osmotic minipumps (Model 1003D Micro-osmotic Pump; Alzet, Cupertino, CA, USA) delivering angiotensin II (1 mg/kg/min) were subcutaneously implanted for 48 hours. In the experimental group, mice received intraperitoneal injections of Ferrostatin-1 (Fer-1; 1 mg/kg/day; Sigma-Aldrich) starting on the 7th day of induction (Fer-1 group, n = 15). Control mice were treated with vehicle (PBS) and BAPN (AD group, n = 15). Histological analysis Excised aortic tissues were fixed in 4% formaldehyde overnight, dehydrated, embedded in paraffin, and sectioned into 4 µm slices. These sections were stained using hematoxylin and eosin (H&E), Masson's trichrome, or elastin Verhoeff-van Gieson (EVG) staining techniques and examined under a light microscope. RNA sequencing The purified RNA was used for the preparation of the sequencing library by TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer’s recommendations. Total RNA (1 µg) was purified to isolate mRNA using oligo (dT)-coupled magnetic beads. Fragmented mRNA was used to synthesize first-strand cDNA via reverse transcription with random primers. Double-stranded cDNA was generated, and 3’ ends were adenylated before adaptor ligation and purification using the AMPure XP system (Beckman Coulter, Beverly, USA). Libraries were assessed for quality using the Agilent Bioanalyzer 2100 system and real-time PCR, then sequenced on an Illumina NovaSeq 6000 platform (150 bp paired-end reads) by Genomics, BioSci & Tech Co. (New Taipei City, Taiwan). Bioinformatics and network analysis Bioinformatics analyses were conducted using the Ingenuity Pathway Analysis (IPA) software (QIAGEN, Redwood City, CA, USA), focusing on canonical pathways, diseases and functions, regulatory effects, upstream regulators, and molecular networks. Additional analyses, including hierarchical clustering (heatmap), principal component analysis (PCA) and volcano plots were performed using R packages integrated with the standalone version of Integrated Differential Expression and Pathway Analysis (iDEP) version 2.0 ( http://bioinformatics.sdstate.edu/idep/ ). Statistical analysis Statistical analyses were performed using the IPA platform and other methods. Pearson’s correlation was employed to assess group correlations. Differentially expressed genes (DEGs) were analyzed using a two-tailed unpaired t-test or Fisher's exact test, with significance thresholds set at fold change (FC) ≥ 1.2 and p < 0.05. For IPA, results with |z-score| ≥ 2 and/or overlap p-value < 0.05 were considered statistically significant. Results Treatment with Fer-1 attenuated AD development in mice Mice treated with Fer-1 showed a marked reduction in the development and progression of AD. Histological analysis further supported these findings, showing preserved aortic wall integrity and reduced structural damage in Fer-1-treated mice (Figure 1A). This therapeutic effect was evident in both the decreased incidence of AD (Figure 1B) and improved survival rates (Figure 1C) compared to the untreated AD group. These results suggest that Fer-1 plays a protective role in AD development. Identification of differentially expressed genes (DEGs) after Fer-1 treatment in AD mice To evaluate the consistency of gene expression among the samples, a correlation heatmap was generated, revealing strong positive correlations with no outlier samples (Figure 2A). Principal component analysis (PCA) further supported these findings, showing a clear separation between Fer-1-treated and AD control groups. Samples within each group formed distinct clusters, indicating high reproducibility and consistency in gene expression patterns (Figure 2B). 922 DEGs were identified when comparing Fer-1-treated aortic samples to AD control samples. Of these, 416 genes were upregulated, while 506 were downregulated (Figures 2C and 2D). The top 10 upregulated and downregulated genes are summarized in Table 1, highlighting key transcriptional changes induced by Fer-1 treatment. These findings will provide a comprehensive overview of the transcriptional alterations associated with Fer-1 treatment and offer a foundation for further investigation into the molecular mechanisms underlying its protective effects in AD. Upstream regulators of differential gene expression Upstream regulators, including transcription factors, cytokines, small RNAs, receptors, kinases, chemical molecules, and pharmacological agents, influence gene expression patterns. Using a threshold of overlap p-value 2), and 270 were significantly inhibited (activation Z-score < −2). The top 15 activated and inhibited upstream regulators are listed in Table 2. MEF2C was identified as the most potent inhibitor (Z-score = −4.358; overlap p-value = 3.06 × 10 -1 8), regulating 29 enriched target genes, including ABRA, ACTA1, ACTN2, ATP2A1, Ccl9, CKM, COL10A1, CXCL2, CXCL6, FOSB, IL6, ITGB1BP2, KCNA5, KCNJ2, LMOD2, MMP8, MYH1, MYH7, MYL11, MYL2, MYOM2, MYOT, MYOZ1, PPARGC1A, SMYD1, TNNI1, TNNI2, TNNT1, TTN (Figure 3A). In contrast, KDM5A emerged as the most robust activator (Z-score = 3.441; overlap p-value = 3.71 × 10-9), targeting 18 genes, including ACTN2 ,Actn3, CACNA1S, HMOX1, HOMER1, MYBPC1, MYH2, MYH4, MYH7, MYOM2, PGAM2, REEP1, RYR1, SOD2, TCAP, TNNC2, TNNI2, TNNT1, TRIM72 (Figure 3B). In-depth analysis of DEGs revealed that MEF2C primarily regulates genes associated with vascular cell functions and vascular development (Figure 3C). KDM5A modulated genes involved in cellular homeostasis (Figure 3D). Additionally, NR5A2 was linked to the regulation of lipid metabolism (Figure 3E). Differential miRNA expression analysis identified 122 miRNAs with altered expression in the Fer-1-treated group compared to the AD group (Table S1). Using an absolute Z-score > 2 as the threshold, the most significantly upregulated and downregulated miRNAs were highlighted. Their regulatory networks, including target genes, are illustrated in Figure 4, providing insights into their roles in modulating the observed transcriptional changes. These findings underscore the complex regulatory mechanisms driving the effects of Fer-1 treatment, particularly in vascular development, homeostasis, and lipid metabolism. Functional prediction and enriched canonical pathways To understand the biological implications of DEGs, we employed multiple analytical approaches by IPA software for comprehensive canonical pathway investigation. IPA revealed 145 enriched canonical pathways under the significance threshold of –log(p-value) > 1.3, with 72 pathways meeting the additional criteria of –log(p-value) > 1.3 and |z-score| ≥ 0.0 as shown in Table S2. By applying a significance threshold of an absolute z-score greater than 2.0, we identified several key findings s sown in Figure 5. Pathways such as ‘Dilated Cardiomyopathy Signaling Pathway’ (z-score = 3.873), the ‘Synaptogenesis Signaling Pathway’ (z-score = 2.840), and the ‘CREB Signaling in Neurons’ (z-score = 2.132) showed significant activation. Pathways like ‘SPINK1 Pancreatic Cancer Pathway’ (z-score = −3.317), the ‘ILK Signaling’ (z-score =−3.051), the ‘Tumor Microenvironment Pathway’ (z-score = −2.333), the ‘Actin Cytoskeleton Signaling’ (z-score = −2.138), the ‘Semaphorin Neuronal Repulsive Signaling Pathway’ (z-score = −2.121) and ‘Role of IL-17F in Allergic Inflammatory Airway Diseases’ (z-score = −2.000) demonstrated significant inhibition. Additional specific signaling pathways associated with AD, including‘IL-17’ ‘ferroptosis’, ‘cytoskeleton signaling’ and ‘macrophage classical activation signaling pathways’ were shown in Figure S1-S4. These analyses provide valuable insights into the complex biological functions and pathways modulated by Fer-1, shedding light on its potential therapeutic mechanisms. Diseases and Biofunction Analysis Using the IPA system with a significance threshold of –log(P‑value) > 4, the role of Fer-1 in disease and cellular functions was analyzed. The analysis revealed key classifications of disease and disorders, molecular and cellular functions, and physiological system development and functions, summarized in a histogram (Figure 6). Fer-1 was found to play significant roles in cellular functions relevant to AD, particularly in processes associated with inflammation and vascular dynamics. Notable functions modulated by Fer-1 included the "Response of phagocytes" (–log(P‑value) = 5.769, Z‑score = –1.836), indicating an inhibitory effect on phagocyte-related responses. Similarly, Fer-1 was linked to the "Occlusion of blood vessels" (–log(P‑value) = 5.635, Z‑score = –1.93), further suggesting its regulatory impact on vascular integrity. Additional associations were identified with "Inflammation of organs" (–log(P‑value) = 10.578, Z‑score = –0.316) and the "Synthesis of reactive oxygen species" (–log(P‑value) = 6.142, Z‑score = –0.087), processes integral to oxidative stress and inflammatory responses (Figure S5–S8). These findings highlight Fer-1's multifaceted role in modulating cellular responses related to AD, emphasizing its potential as a therapeutic agent targeting inflammatory and vascular dysfunctions. Interaction network analysis The interaction network analysis identified key molecular interactions within the dataset, providing insights into functional relationships. Networks were ranked based on score values, with detailed results provided in Table S3. The highest-ranked network, with a score of 33, was primarily associated with "Cardiovascular Disease, Cell Death and Survival, and Cellular Assembly and Organization." This network included 21 molecules from the DEGs dataset: ACACA, ASB5, BPGM, C11orf54, Ces2c, CLVS2, CLYBL, CXCR3, DISP2, DZANK1, ENO1, ENO2, EYA1, GIPC2, Gypa, GZMA, HEMGN, HNF4A, IFI16, IGF2BP2, and Ldh. The interactions among these 21 DEGs are visualized in Figure 7, illustrating their complex relationships and potential cooperative roles in cardiovascular disease mechanisms, cell survival processes, and cellular organization. These findings underscore the importance of these molecular networks in the context of Fer-1 treatment and its therapeutic implications. Discussion Ferroptosis, characterized by iron-dependent lipid peroxidation, has emerged as a central mechanism in vascular injury. The histological analyses in this study demonstrated that Fer-1 preserved aortic wall integrity in a BAPN/Ang-II-induced AD mouse model. The observed reduction in AD incidence and mortality highlights the critical role of ferroptosis in early structural damage and disease progression. These findings align with previous reports indicating that increased oxidative stress and inflammation create a favorable environment for ferroptosis in AD ( 12 , 14 , 17 ). Gene expression analysis identified key upstream regulatory molecules influenced by Fer-1 treatment. MEF2C and KDM5A were identified as potential regulators of AD pathogenesis. MEF2C is known to protect against atherosclerosis by inhibiting TLR/NF-κB activation, SMC migration ( 18 ), and proliferation ( 19 , 20 ). It regulates KLF2, which enhances endothelial barrier function and inhibits atherosclerosis, thrombosis, and coronary artery lesions ( 18 , 21 , 22 ). Hyper uric acid exerts thrombogenic effects in mice by up-regulating let-7c and activating the NF-κB pathway in a MEF2C-dependent manner ( 23 ). MEF2C overexpression mitigates apoptosis in cerebral ischemia preconditioning and suppresses inflammation and oxidative stress by inhibiting NF-κB phosphorylation ( 24 , 25 ). Additionally, MEF2C alleviates postoperative cognitive dysfunction by repressing ferroptosis ( 26 ). KDM5A, another identified regulator, maintains genomic integrity and modulates key processes such as cell cycle, apoptosis, and metabolism ( 27 , 28 ). KDM5A plays a pivotal role in vascular VSMC homeostasis, regulating proliferation, differentiation, and vascular remodeling. Loss of KDM5A function disrupts VSMC homeostasis, increasing susceptibility to AD ( 29 ). Additionally, another upstream regulator, NR5A2, identified in our datasets, is involved in lipid metabolism. NR5A2 plays critical roles in embryonic development, cholesterol and bile acid homeostasis, and cell proliferation ( 30 ). NR5A2 has also been shown to indirectly regulate the immune system and associated inflammatory processes via the synthesis of immunoregulatory glucocorticoids in the intestinal crypts ( 31 ). Tissue-specific deletion or inhibition of NR5A2 and associated intestinal glucocorticoid synthesis consequently results in increased susceptibility to the development of intestinal inflammatory disorders ( 32 ). Based on our analysis, the upstream regulators MEF2C, KDM5A, and NR5A2, along with the downregulation of their target genes (Ccl9, COL10A1, CXCL2, MMP8, Ccl7, IL1B, IL6, SOD2, and others), may represent an additional mechanism by which Fer-1 exerts its therapeutic effects in AD. This mechanism likely involves the modulation of immune responses, oxidative stress, apoptosis, and lipid metabolism, as well as the prevention of inappropriate phenotypic alterations in SMCs and the enhancement of endothelial barrier function. In addition to epigenetic regulation, miRNAs were also implicated in the protective effects of Fer-1. MiR-361-5p, previously associated with acute coronary syndrome and endothelial cell function ( 33 , 34 ). The expression of miR-361-5p was significantly decreased in ox-LDL injured vascular SMCs, while lncRNA MEG3-derived miR-361-5p regulate vascular SMC proliferation and apoptosis by targeting ABCA1( 35 ). In this study, miR-361-5p was predicted to be upregulated following Fer-1 treatment. It was identified as an upstream regulator of genes such as HMOX1, ATP1B4, CACNG1, G6PC1, and HOMER1, which are involved in modulating oxidative stress, inflammation, and ion homeostasis. Previous research has established a strong association between HMOX1 and ferroptosis ( 33 , 36 , 37 ) ), with excessive HMOX1 expression potentially triggering ferroptosis. Conversely, miR-3151-5p expression was found to be downregulated in Fer-1-treated mice. This miRNA is predicted to target genes related to cell death (e.g., CASP3, CASP9, FAS), ion homeostasis (e.g., CALB1), and inflammation (e.g., IL6, IL1B, NLRP3). Notably, the downregulation of IL6 and IL1B, key inflammatory mediators, observed in Fer-1-treated mice corresponds with the reduction in inflammation, further supporting the therapeutic effects of Fer-1. The plasticity of SMCs is crucial for vascular compliance and ECM regulation, particularly in aortic disease pathogenesis. PAI-1 inhibits cofilin, a key cytoskeletal regulator, thereby influencing SMC stiffness and F-actin content ( 38 ). Consistent with our findings, Fer-1 treatment regulated cytoskeleton polymerization by reducing F-actin level (Figure S3 ). ILK an intracellular serine/threonine kinase, plays a critical role in cell-matrix interactions and signal transduction ( 39 ). It induces AKT phosphorylation and p21 degradation, which are associated with SMC migration and proliferation ( 40 ). Furthermore, ILK activates MMP-9 promoter, and consequently MMP-9 expression through the GSK-3β/AP-1 pathway ( 41 ) and contributes to Ang-II-induced renal inflammation ( 42 ). IL-17 plays a critical role in acute inflammation and is reported to participate in AD pathogenesis ( 43 ) by interfering with TGF-β signal and altering ECM metabolism ( 44 ). Additionally, Fer-1 treatment downregulated pro-inflammatory M1 macrophage pathways while upregulating anti-inflammatory M2 macrophage pathways (Figure S4 ). Findings form our datasets according enrichment analysis on canonical pathways of IPA suggest that Fer-1 preserves SMC differentiation and attenuates ECM remodeling through its anti-inflammatory and cytoskeletal regulatory effects. Gene interaction network analysis further revealed key molecules such as CXCR3, ACACA, and BPGM, which play roles in inflammation, lipid metabolism, and vascular wall integrity. CXCR3 regulates immune cell recruitment and inflammatory signaling, processes exacerbating aortic wall damage. ACACA, a key enzyme in fatty acid biosynthesis, influences lipid homeostasis, while BPGM reflects metabolic adaptation under oxidative stress. These interactions indicate that Fer-1 not only inhibits ferroptosis but also modulates broader metabolic and inflammatory pathways to support vascular health. Other insightful pathway in this study identified 922 DEGs associated with ferroptosis inhibition, including pathways related to phagocytes response, occlusion of blood vessel, and ROS synthesis to against AD (Figure S5 - S6 ). While this research provides compelling evidence for the role of ferroptosis in AD and the protective effects of Fer-1, certain limitations remain. Functional validation of identified DEGs and their roles in ferroptosis and AD pathogenesis is necessary. Additionally, transgenic animal studies could clarify the contributions of targets such as MEF2C and KDM5A. The acute effects of Fer-1 were the primary focus; future studies should investigate its long-term efficacy and potential side effects. Dose-response studies are also needed to optimize therapeutic outcomes. Conclusion This research elucidates the mechanism of ferroptosis in AD pathogenesis and establishes Fer-1 as a promising therapeutic intervention. By attenuating ferroptosis-induced lipid peroxidation, oxidative stress, and inflammation, Fer-1 effectively preserves aortic wall integrity and potentially reduces AD incidence and mortality. The findings highlight the therapeutic potential of ferroptosis-targeted interventions and provide a foundation for future translational research. Abbreviations AD Aortic Dissection Fer-1 Ferrostatin-1 SMC Smooth Muscle Cell ECM Extracellular Matrix BAPN β-aminopropionitrile Ang-II Angiotensin II SPF Specific Pathogen-free IPA Ingenuity Pathway Analysis PCA Principal Component Analysis DEGs Differentially Expressed Genes FC Fold Change Declarations Funding This work was financially supported by the Wan Fang Hospital, Taipei Medical University (112-wf-eva-23) and the National Science and Technology Council, Taiwan (NSTC 112-2314-B-038 -122 -MY3). Authorship contributions Chun-Che Shih*: writing – original draft, writing – review and editing, data curation. Chi-Yu Chen *: writing – original draft, writing – review and editing, formal analysis, methodology. Chih-Pin Chuu: formal analysis, writing – review and editing, visualization. Chun-Yang, Huang: formal analysis, writing – review and editing. Chia-Jung Lu: methodology, writing – review and editing. Hsin-Ying Lu: conceptualization, writing – review and editing, funding acquisition, project administration. *These authors contributed equally to this work. Availability of data All data presented in the present study are available from the corresponding author on reasonable request. Declarations Competing interests The authors declare no competing interests. Ethical approval All animal experiments were conducted with the approval of Laboratory Animal Center of Taipei Medical University (Protocol number: No: LAC-2022-0469). Consent for publication Not applicable. References Nienaber CA, Clough RE, Sakalihasan N et al (2016) Aortic dissection. Nat Rev Dis Primers 2:16053 Evangelista A, Isselbacher EM, Bossone E et al (2018) Insights From the International Registry of Acute Aortic Dissection: A 20-Year Experience of Collaborative Clinical Research. Circulation 137:1846–1860 Shen YH, LeMaire SA, Webb NR, Cassis LA, Daugherty A, Lu HS (2020) Aortic Aneurysms and Dissections Series. 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Arterioscler Thromb Vasc Biol 40:189–205 Tables Table 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx S1IL17pathway.tif Figure S1: IL17 pathway S2ferroptosissignalingpathway.tif Figure S2: Ferroptosis signaling pathway S3actincytoskeletonsignaling.tif Figure S3: Actin cytoskeleton signaling S4macrophageclassicalactivationsignalingpathway.tif Figure S4 macrophage classical activation signaling pathway S5responseofphagocytes.tif Figure S5 response of phagocytes S6Occlusionofbloodvessel.tif Figure S6 Occlusion of blood vessel S7inflammationoforgan.tif Figure S7 inflammation of organ S8synthesisofROS.tif Figure S8 synthesis of ROS TableS1SummaryofupstreamanalysisformiRNA.xls Table S1: Table S1 Summary of upstream analysis for differentially expressed Fer-1-related miRNA in AD aorta TableS2Summaryofenrichedcanonicalpathways.xls Table S2 Summary of differentially expressed Fer-1-related genes enriched canonical pathways in AD aorta TableS3Summaryofenrichedmolecularnetworkanalysis.xls Table S3 Summary of enriched molecular network analysis for differentially expressed Fer-1-related genes in AD aorta 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. 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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-6061217","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":417941592,"identity":"c2542134-0828-474f-9e15-506e9f782708","order_by":0,"name":"Chun-Che Shih","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chun-Che","middleName":"","lastName":"Shih","suffix":""},{"id":417941595,"identity":"09ac6698-98cd-48d7-93e3-7be40791d117","order_by":1,"name":"Chi-Yu Chen","email":"","orcid":"","institution":"University of Pittsburgh School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chi-Yu","middleName":"","lastName":"Chen","suffix":""},{"id":417941601,"identity":"941c4393-0a76-47e0-bfc5-cf516474807f","order_by":2,"name":"Chih-Pin Chuu","email":"","orcid":"","institution":"National Health Research Institutes, Miaoli County","correspondingAuthor":false,"prefix":"","firstName":"Chih-Pin","middleName":"","lastName":"Chuu","suffix":""},{"id":417941603,"identity":"ef3ebdab-4c67-48be-9e50-c16899be6d7a","order_by":3,"name":"Chun-Yang Huang","email":"","orcid":"","institution":"National Yang-Ming Chiao-Tung University","correspondingAuthor":false,"prefix":"","firstName":"Chun-Yang","middleName":"","lastName":"Huang","suffix":""},{"id":417941604,"identity":"308b06cf-0f19-47b9-a61b-a93fac6ef946","order_by":4,"name":"Chia-Jung Lu","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chia-Jung","middleName":"","lastName":"Lu","suffix":""},{"id":417941605,"identity":"4397881e-d6c5-49a0-b001-847d32357e9b","order_by":5,"name":"Hsin-Ying Lu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYBACAwbGBhDJwAdkHEiogIhKEKWFjYG58cCHMwYwLQZ4tEABGwN788GZbURoMec/3PaYp+COXZtEYsNh3nl/og0OMB+8zcPwJ7EBhxbLGYntxjwGz5IhWrYZ5G44wJZszcNggFOLwQ3GNmkeg8PJbAgtPGbSQC25OLWcP4isZQ5IC/83/FoOJIK12IG0HJzZALaFDb+WG4ltknMMDiew8TxsOPDhmHHuzMNsxpZzDIzrcTvs+DOJN38O2/Ozpz/+kFAjl9t3vPnhjTcVcsY4dIABEw8DA1L4MIONwqeBgYHxBwODPX4lo2AUjIJRMKIBAIV2XASAqixfAAAAAElFTkSuQmCC","orcid":"","institution":"Taipei Medical University","correspondingAuthor":true,"prefix":"","firstName":"Hsin-Ying","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2025-02-19 06:38:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6061217/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6061217/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76868977,"identity":"acf4d96c-12c9-4056-ad37-d08ae53cc51c","added_by":"auto","created_at":"2025-02-21 15:07:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2257496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFer-1 ameliorates the etiopathology of BAPN/Ang-II-induced AD in mice. \u003c/strong\u003e(A) Representative morphologies of aortas, H\u0026amp;E and VVG staining in aorta. (B) Incidence. (C) Mortality.\u003c/p\u003e","description":"","filename":"F1.png","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/001d894e9f6135453ea7a896.png"},{"id":76868975,"identity":"f45cf3c3-4c51-4b95-a005-d842e52762a2","added_by":"auto","created_at":"2025-02-21 15:07:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":528599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed genes (DEGs) in Fer-1- treated aorta compared with AD controls.\u003c/strong\u003e (A) The correlation of heatmap among the samples. (B) Principal component analysis (PCA) of RNA-seq datase (C) volcano plot displaying DEGs under the cut-off: p value \u0026lt; 0.05 and absolute log2FoldChange \u0026gt;0.58. Green dots: significantly downregulated genes; blue dots: significantly upregulated genes. (D) Heatmap of DEGs.\u003c/p\u003e","description":"","filename":"F2.png","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/b25c95690007a4c7a9e1ad7d.png"},{"id":76870558,"identity":"ace57b7c-2f8d-48fc-a010-2a5871ad9e1f","added_by":"auto","created_at":"2025-02-21 15:23:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":670799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUpstream regulator analysis of DEGs\u003c/strong\u003e. (A) Downstream targets of MEF2C among the DEGs. (B) Downstream targets of KDM5A among the DEGs. (C) The diseases and biofunctions were regulated by MEF2C. (D) The diseases and biofunctions were regulated by KDM5A. (E) The diseases and biofunctions were regulated by NR5A2.\u003c/p\u003e","description":"","filename":"F3.png","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/ca0a50546d91922b1e9630a6.png"},{"id":76870180,"identity":"5c41703d-8951-47f0-8866-9e1b9a246dd8","added_by":"auto","created_at":"2025-02-21 15:15:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1312431,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegrated pathway analysis of upstream molecules, gene expression and function predictions. \u003c/strong\u003e(A) miR-361, (B) miR-3151-5p. The top row shows each miRNA, the middle row shows the gene predicted to be regulated by each miRNA and the bottom row shows the function predicted to be involved. Orange miRNAs are upregulated, blue miRNAs are downregulated, peach genes are upregulated, green genes are downregulated and orange predicted functions are upregulated.\u003c/p\u003e","description":"","filename":"F4.png","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/67685b54c468955912ee3bfc.png"},{"id":76870182,"identity":"47ecc1d7-d06a-465b-aa59-69c9e15d9fdc","added_by":"auto","created_at":"2025-02-21 15:15:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105369,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnriched canonical pathways of DEGs.\u003c/strong\u003e Blue band: the negative prediction of the pathway; orange band: the active prediction of the pathway. The filter was absolute z-score \u0026gt; 2.0. The threshold line was drawn at -log(p-value) = 1.3.\u003c/p\u003e","description":"","filename":"F5.png","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/91d61bfad3da34209ed91504.png"},{"id":76868984,"identity":"8fd1566e-de1a-4e8a-8da2-2745fe528c4c","added_by":"auto","created_at":"2025-02-21 15:07:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":74060,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnriched diseases and bio functions of DEGs. \u003c/strong\u003e10 representative classification of diseases and functions possibly mediated by Fer-1 are plotted. The threshold line was drawn at -log(p-value) = 1.3.\u003c/p\u003e","description":"","filename":"F6.png","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/944f72f1771115f9548271f1.png"},{"id":76870559,"identity":"c7106e8e-2be2-48d7-a419-c16d4130febd","added_by":"auto","created_at":"2025-02-21 15:23:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":588362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene interaction network map.\u003c/strong\u003eThis network consists of the top ranked network found associated with the role of Fer-1 treatment in cardiovascular disease, cell death and survival, cellular assembly and organization.\u003c/p\u003e","description":"","filename":"F7.png","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/21f83fbc249a5986fe577a34.png"},{"id":76972943,"identity":"91660407-bbe2-4f6f-9f65-db7939c8b8c3","added_by":"auto","created_at":"2025-02-23 19:01:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5696867,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/3c54bd4c-ce16-4598-8027-0c4ee8b492cd.pdf"},{"id":76868978,"identity":"4a6dbd68-a1e5-4e97-b544-56adc22c131c","added_by":"auto","created_at":"2025-02-21 15:07:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30778,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/48665a163bc6f12a54bfacbb.docx"},{"id":76869012,"identity":"37930ea4-176d-4616-95da-1773f447f366","added_by":"auto","created_at":"2025-02-21 15:07:10","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":49754620,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1: IL17 pathway\u003c/p\u003e","description":"","filename":"S1IL17pathway.tif","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/848dccb807c861646b1811e6.tif"},{"id":76870184,"identity":"010ca09a-5fb9-42c5-8cca-53acfcbd0f6f","added_by":"auto","created_at":"2025-02-21 15:15:09","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5740560,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2: Ferroptosis signaling pathway\u003c/p\u003e","description":"","filename":"S2ferroptosissignalingpathway.tif","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/8e2016d4171d2ee9c5bf0af6.tif"},{"id":76870187,"identity":"2e6d90fb-76b8-4d69-8d1a-15863ea1e686","added_by":"auto","created_at":"2025-02-21 15:15:09","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4538968,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S3: Actin cytoskeleton signaling\u003c/p\u003e","description":"","filename":"S3actincytoskeletonsignaling.tif","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/8bafd28a94b97e416b3de643.tif"},{"id":76868993,"identity":"c31aab1b-230d-40c0-8a52-f03c7dc4e554","added_by":"auto","created_at":"2025-02-21 15:07:09","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12764080,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S4 macrophage classical activation signaling pathway\u003c/p\u003e","description":"","filename":"S4macrophageclassicalactivationsignalingpathway.tif","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/aecfc49cd218f729443287bc.tif"},{"id":76870183,"identity":"4e926321-df6a-436c-a74f-fc819c57130b","added_by":"auto","created_at":"2025-02-21 15:15:09","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1538704,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S5 response of phagocytes\u003c/p\u003e","description":"","filename":"S5responseofphagocytes.tif","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/e215506ef89ba044ef81fd59.tif"},{"id":76868991,"identity":"8b71554c-2364-4a2b-9f69-3de852d46881","added_by":"auto","created_at":"2025-02-21 15:07:09","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1443132,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S6 Occlusion of blood vessel\u003c/p\u003e","description":"","filename":"S6Occlusionofbloodvessel.tif","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/ade90ad9d4aa30162a34794d.tif"},{"id":76869006,"identity":"aa9b2861-c152-406a-91be-55c9a7f82c55","added_by":"auto","created_at":"2025-02-21 15:07:09","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":1765952,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S7 inflammation of organ\u003c/p\u003e","description":"","filename":"S7inflammationoforgan.tif","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/0413b0725a45c5d76ebf8d8e.tif"},{"id":76868996,"identity":"cdaab46d-b12e-4f53-ab45-bc0a046cb768","added_by":"auto","created_at":"2025-02-21 15:07:09","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1755108,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S8 synthesis of ROS\u003c/p\u003e","description":"","filename":"S8synthesisofROS.tif","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/3c379682690a156e47bcde9b.tif"},{"id":76870191,"identity":"7ff7ca80-88bc-4873-a701-210d706b3feb","added_by":"auto","created_at":"2025-02-21 15:15:09","extension":"xls","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":64000,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1: Table S1 Summary of upstream analysis for differentially expressed Fer-1-related miRNA in AD aorta\u003c/p\u003e","description":"","filename":"TableS1SummaryofupstreamanalysisformiRNA.xls","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/4124c96cc49ebb8522f0cb93.xls"},{"id":76868986,"identity":"c7fadf1e-9772-4922-99ef-255a1cc6b68b","added_by":"auto","created_at":"2025-02-21 15:07:09","extension":"xls","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":59904,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2 Summary of differentially expressed Fer-1-related genes enriched canonical pathways in AD aorta\u003c/p\u003e","description":"","filename":"TableS2Summaryofenrichedcanonicalpathways.xls","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/7a3e08e7fbe195fb0e643eae.xls"},{"id":76869010,"identity":"8cb34a1d-6179-4601-94fa-6767963a7a88","added_by":"auto","created_at":"2025-02-21 15:07:09","extension":"xls","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":43520,"visible":true,"origin":"","legend":"\u003cp\u003eTable S3 Summary of enriched molecular network analysis for differentially expressed Fer-1-related genes in AD aorta\u003c/p\u003e","description":"","filename":"TableS3Summaryofenrichedmolecularnetworkanalysis.xls","url":"https://assets-eu.researchsquare.com/files/rs-6061217/v1/6a47713c5e59e163b2c11121.xls"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptome Insights into Protective Mechanisms of Ferroptosis Inhibition in Aortic Dissection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAortic dissection (AD) is a life-threatening vascular condition characterized by the separation of the aortic wall layers, resulting in severe complications such as rupture and organ ischemia (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Despite significant advancements in diagnostic imaging and surgical management, the limited understanding of AD pathogenesis hampers the development of effective early-stage intervention strategies (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Recent studies have identified oxidative stress, inflammation, smooth muscle cell (SMC) phenotypic transition and extracellular matrix (ECM) degradation as critical contributors to AD progression (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). These pathological processes frequently culminate in cell death, further compromising the structural integrity of the aortic wall (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Although certain agents targeting apoptosis or autophagic cell death have demonstrated protective effects in mouse models of AD, their clinical translation has yet to be achieved. These findings emphasize the pivotal role of regulated cell death in AD pathogenesis and highlight the potential of targeting these pathways as a promising therapeutic strategy (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the various forms of regulated cell death, ferroptosis\u0026mdash;an iron-dependent mechanism characterized by lipid peroxidation\u0026mdash;has emerged as a critical factor in vascular injury and inflammation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Ferroptosis has been implicated in the pathogenesis of several cardiovascular diseases, including doxorubicin-induced cardiotoxicity (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), ischemia-reperfusion injury (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), heart failure (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), AD (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) and stroke (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In AD, elevated oxidative stress creates an environment conducive to ferroptotic cell death, which may exacerbate structural damage to the aortic wall (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). However, the interplay between ferroptosis and other pathological processes in AD, such as smooth muscle cell dysfunction and ECM degradation, remains inadequately explored (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). This knowledge gap underscores the critical need for focused investigations into ferroptosis-mediated mechanisms and their potential therapeutic modulation in AD.\u003c/p\u003e \u003cp\u003eTo have better understand the therapeutic potentials of targeting ferroptosis, ferrostatin-1 (Fer-1), we have examined the effects of Fer-1, a potent ferroptosis inhibitor, in β-aminopropionitrile (BAPN) and angiotensin II (Ang-II)-induced AD mouse models. Fer-1 has demonstrated efficacy in preclinical models of oxidative stress-related diseases by scavenging lipid peroxides and preserving cellular antioxidant defenses. These mechanisms effectively mitigate oxidative damage and inflammation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). This study aims to elucidate the molecular mechanisms underlying the therapeutic effects of Fer-1 and its role in inhibiting ferroptosis through histological, transcriptomic, and pathway analyses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eModel establishment and interventions\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee of Taipei Medical University. Male C57BL/6J mice were housed in a specific pathogen-free (SPF) facility under a 12-hour light-dark cycle and fed a standard diet. An aortic dissection (AD) model was induced in three-week-old mice by oral administration of 0.5% β-aminopropionitrile (BAPN; Sigma-Aldrich, St. Louis, MO, USA) for four weeks. Subsequently, osmotic minipumps (Model 1003D Micro-osmotic Pump; Alzet, Cupertino, CA, USA) delivering angiotensin II (1 mg/kg/min) were subcutaneously implanted for 48 hours. In the experimental group, mice received intraperitoneal injections of Ferrostatin-1 (Fer-1; 1 mg/kg/day; Sigma-Aldrich) starting on the 7th day of induction (Fer-1 group, n\u0026thinsp;=\u0026thinsp;15). Control mice were treated with vehicle (PBS) and BAPN (AD group, n\u0026thinsp;=\u0026thinsp;15).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistological analysis\u003c/h3\u003e\n\u003cp\u003eExcised aortic tissues were fixed in 4% formaldehyde overnight, dehydrated, embedded in paraffin, and sectioned into 4 \u0026micro;m slices. These sections were stained using hematoxylin and eosin (H\u0026amp;E), Masson's trichrome, or elastin Verhoeff-van Gieson (EVG) staining techniques and examined under a light microscope.\u003c/p\u003e\n\u003ch3\u003eRNA sequencing\u003c/h3\u003e\n\u003cp\u003eThe purified RNA was used for the preparation of the sequencing library by TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer\u0026rsquo;s recommendations. Total RNA (1 \u0026micro;g) was purified to isolate mRNA using oligo (dT)-coupled magnetic beads. Fragmented mRNA was used to synthesize first-strand cDNA via reverse transcription with random primers. Double-stranded cDNA was generated, and 3\u0026rsquo; ends were adenylated before adaptor ligation and purification using the AMPure XP system (Beckman Coulter, Beverly, USA). Libraries were assessed for quality using the Agilent Bioanalyzer 2100 system and real-time PCR, then sequenced on an Illumina NovaSeq 6000 platform (150 bp paired-end reads) by Genomics, BioSci \u0026amp; Tech Co. (New Taipei City, Taiwan).\u003c/p\u003e\n\u003ch3\u003eBioinformatics and network analysis\u003c/h3\u003e\n\u003cp\u003eBioinformatics analyses were conducted using the Ingenuity Pathway Analysis (IPA) software (QIAGEN, Redwood City, CA, USA), focusing on canonical pathways, diseases and functions, regulatory effects, upstream regulators, and molecular networks. Additional analyses, including hierarchical clustering (heatmap), principal component analysis (PCA) and volcano plots were performed using R packages integrated with the standalone version of Integrated Differential Expression and Pathway Analysis (iDEP) version 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/idep/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/idep/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using the IPA platform and other methods. Pearson\u0026rsquo;s correlation was employed to assess group correlations. Differentially expressed genes (DEGs) were analyzed using a two-tailed unpaired t-test or Fisher's exact test, with significance thresholds set at fold change (FC)\u0026thinsp;\u0026ge;\u0026thinsp;1.2 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. For IPA, results with |z-score| \u0026ge; 2 and/or overlap p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eTreatment with Fer-1 attenuated AD development in mice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice treated with Fer-1 showed a marked reduction in the development and progression of AD. Histological analysis further supported these findings, showing preserved aortic wall integrity and reduced structural damage in Fer-1-treated mice (Figure 1A). This therapeutic effect was evident in both the decreased incidence of AD (Figure 1B) and improved survival rates (Figure 1C) compared to the untreated AD group. These results suggest that Fer-1 plays a protective role in AD development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of differentially expressed genes (DEGs) after Fer-1 treatment in AD mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the consistency of gene expression among the samples, a correlation heatmap was generated, revealing strong positive correlations with no outlier samples (Figure 2A). Principal component analysis (PCA) further supported these findings, showing a clear separation between Fer-1-treated and AD control groups. Samples within each group formed distinct clusters, indicating high reproducibility and consistency in gene expression patterns (Figure 2B).\u003c/p\u003e\n\u003cp\u003e922 DEGs were identified when comparing Fer-1-treated aortic samples to AD control samples. Of these, 416 genes were upregulated, while 506 were downregulated (Figures 2C and 2D). The top 10 upregulated and downregulated genes are summarized in Table 1, highlighting key transcriptional changes induced by Fer-1 treatment.\u003c/p\u003e\n\u003cp\u003eThese findings will provide a comprehensive overview of the transcriptional alterations associated with Fer-1 treatment and offer a foundation for further investigation into the molecular mechanisms underlying its protective effects in AD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUpstream regulators of differential gene expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpstream regulators, including transcription factors, cytokines, small RNAs, receptors, kinases, chemical molecules, and pharmacological agents, influence gene expression patterns. Using a threshold of overlap p-value \u0026lt; 0.05, 1,728 upstream regulators were identified. Among these, 93 were significantly activated (activation Z-score \u0026gt; 2), and 270 were significantly inhibited (activation Z-score \u0026lt; \u0026minus;2). The top 15 activated and inhibited upstream regulators are listed in Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMEF2C was identified as the most potent inhibitor (Z-score = \u0026minus;4.358; overlap p-value = 3.06 \u0026times; 10\u003csup\u003e-1\u003c/sup\u003e8), regulating 29 enriched target genes, including ABRA, ACTA1, ACTN2, ATP2A1, Ccl9, CKM, COL10A1, CXCL2, CXCL6, FOSB, IL6, ITGB1BP2, KCNA5, KCNJ2, LMOD2, MMP8, MYH1, MYH7, MYL11, MYL2, MYOM2, MYOT, MYOZ1, PPARGC1A, SMYD1, TNNI1, TNNI2, TNNT1, TTN (Figure 3A). In contrast, KDM5A emerged as the most robust activator (Z-score = 3.441; overlap p-value = 3.71 \u0026times; 10-9), targeting 18 genes, including ACTN2 ,Actn3, CACNA1S, HMOX1, HOMER1, MYBPC1, MYH2, MYH4, MYH7, MYOM2, PGAM2, REEP1, RYR1, SOD2, TCAP, TNNC2, TNNI2, TNNT1, TRIM72 (Figure 3B). In-depth analysis of DEGs revealed that MEF2C primarily regulates genes associated with vascular cell functions and vascular development (Figure 3C). KDM5A modulated genes involved in cellular homeostasis (Figure 3D). Additionally, NR5A2 was linked to the regulation of lipid metabolism (Figure 3E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDifferential miRNA expression analysis identified 122 miRNAs with altered expression in the Fer-1-treated group compared to the AD group\u0026nbsp;(Table S1). Using an absolute Z-score \u0026gt; 2 as the threshold, the most significantly upregulated and downregulated miRNAs were highlighted. Their regulatory networks, including target genes, are illustrated in Figure 4, providing insights into their roles in modulating the observed transcriptional changes.\u003c/p\u003e\n\u003cp\u003eThese findings underscore the complex regulatory mechanisms driving the effects of Fer-1 treatment, particularly in vascular development, homeostasis, and lipid metabolism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional prediction and enriched canonical pathways\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the biological implications of DEGs, we employed multiple analytical approaches by IPA software for comprehensive canonical pathway investigation.\u003c/p\u003e\n\u003cp\u003eIPA revealed 145 enriched canonical pathways under the significance threshold of \u0026ndash;log(p-value) \u0026gt; 1.3, with 72 pathways meeting the additional criteria of \u0026ndash;log(p-value) \u0026gt; 1.3 and |z-score| \u0026ge; 0.0 as shown in Table S2. By applying a significance threshold of an absolute z-score greater than 2.0, we identified several key findings s sown in Figure 5. Pathways such as \u0026lsquo;Dilated Cardiomyopathy Signaling Pathway\u0026rsquo; (z-score = 3.873), the \u0026lsquo;Synaptogenesis Signaling Pathway\u0026rsquo; (z-score = 2.840), and the \u0026lsquo;CREB Signaling in Neurons\u0026rsquo; (z-score = 2.132) showed significant activation. Pathways like \u0026lsquo;SPINK1 Pancreatic Cancer Pathway\u0026rsquo; (z-score = \u0026minus;3.317), the \u0026lsquo;ILK Signaling\u0026rsquo; (z-score =\u0026minus;3.051), the \u0026lsquo;Tumor Microenvironment Pathway\u0026rsquo; (z-score = \u0026minus;2.333), the \u0026lsquo;Actin Cytoskeleton Signaling\u0026rsquo; (z-score = \u0026minus;2.138), the \u0026lsquo;Semaphorin Neuronal Repulsive Signaling Pathway\u0026rsquo; (z-score = \u0026minus;2.121) and \u0026lsquo;Role of IL-17F in Allergic Inflammatory Airway Diseases\u0026rsquo; (z-score = \u0026minus;2.000) demonstrated significant inhibition. Additional specific signaling pathways associated with AD, including\u0026lsquo;IL-17\u0026rsquo; \u0026lsquo;ferroptosis\u0026rsquo;, \u0026lsquo;cytoskeleton signaling\u0026rsquo; and \u0026lsquo;macrophage classical activation signaling pathways\u0026rsquo; were shown in Figure S1-S4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese analyses provide valuable insights into the complex biological functions and pathways modulated by Fer-1, shedding light on its potential therapeutic mechanisms.\u003c/p\u003e\n\u003ch3\u003eDiseases and Biofunction Analysis\u003c/h3\u003e\n\u003cp\u003eUsing the IPA system with a significance threshold of \u0026ndash;log(P‑value) \u0026gt; 4, the role of Fer-1 in disease and cellular functions was analyzed. The analysis revealed key classifications of disease and disorders, molecular and cellular functions, and physiological system development and functions, summarized in a histogram (Figure 6). Fer-1 was found to play significant roles in cellular functions relevant to AD, particularly in processes associated with inflammation and vascular dynamics. Notable functions modulated by Fer-1 included the \u0026quot;Response of phagocytes\u0026quot; (\u0026ndash;log(P‑value) = 5.769, Z‑score = \u0026ndash;1.836), indicating an inhibitory effect on phagocyte-related responses. Similarly, Fer-1 was linked to the \u0026quot;Occlusion of blood vessels\u0026quot; (\u0026ndash;log(P‑value) = 5.635, Z‑score = \u0026ndash;1.93), further suggesting its regulatory impact on vascular integrity. Additional associations were identified with \u0026quot;Inflammation of organs\u0026quot; (\u0026ndash;log(P‑value) = 10.578, Z‑score = \u0026ndash;0.316) and the \u0026quot;Synthesis of reactive oxygen species\u0026quot; (\u0026ndash;log(P‑value) = 6.142, Z‑score = \u0026ndash;0.087), processes integral to oxidative stress and inflammatory responses (Figure S5\u0026ndash;S8).\u003c/p\u003e\n\u003cp\u003eThese findings highlight Fer-1\u0026apos;s multifaceted role in modulating cellular responses related to AD, emphasizing its potential as a therapeutic agent targeting inflammatory and vascular dysfunctions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInteraction network analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe interaction network analysis identified key molecular interactions within the dataset, providing insights into functional relationships. Networks were ranked based on score values, with detailed results provided in Table S3. The highest-ranked network, with a score of 33, was primarily associated with \u0026quot;Cardiovascular Disease, Cell Death and Survival, and Cellular Assembly and Organization.\u0026quot; This network included 21 molecules from the DEGs dataset: ACACA, ASB5, BPGM, C11orf54, Ces2c, CLVS2, CLYBL, CXCR3, DISP2, DZANK1, ENO1, ENO2, EYA1, GIPC2, Gypa, GZMA, HEMGN, HNF4A, IFI16, IGF2BP2, and Ldh.\u003c/p\u003e\n\u003cp\u003eThe interactions among these 21 DEGs are visualized in Figure 7, illustrating their complex relationships and potential cooperative roles in cardiovascular disease mechanisms, cell survival processes, and cellular organization. These findings underscore the importance of these molecular networks in the context of Fer-1 treatment and its therapeutic implications.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFerroptosis, characterized by iron-dependent lipid peroxidation, has emerged as a central mechanism in vascular injury. The histological analyses in this study demonstrated that Fer-1 preserved aortic wall integrity in a BAPN/Ang-II-induced AD mouse model. The observed reduction in AD incidence and mortality highlights the critical role of ferroptosis in early structural damage and disease progression. These findings align with previous reports indicating that increased oxidative stress and inflammation create a favorable environment for ferroptosis in AD (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGene expression analysis identified key upstream regulatory molecules influenced by Fer-1 treatment. MEF2C and KDM5A were identified as potential regulators of AD pathogenesis. MEF2C is known to protect against atherosclerosis by inhibiting TLR/NF-κB activation, SMC migration (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), and proliferation (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). It regulates KLF2, which enhances endothelial barrier function and inhibits atherosclerosis, thrombosis, and coronary artery lesions (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Hyper uric acid exerts thrombogenic effects in mice by up-regulating let-7c and activating the NF-κB pathway in a MEF2C-dependent manner (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). MEF2C overexpression mitigates apoptosis in cerebral ischemia preconditioning and suppresses inflammation and oxidative stress by inhibiting NF-κB phosphorylation (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Additionally, MEF2C alleviates postoperative cognitive dysfunction by repressing ferroptosis (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). KDM5A, another identified regulator, maintains genomic integrity and modulates key processes such as cell cycle, apoptosis, and metabolism (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). KDM5A plays a pivotal role in vascular VSMC homeostasis, regulating proliferation, differentiation, and vascular remodeling. Loss of KDM5A function disrupts VSMC homeostasis, increasing susceptibility to AD (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Additionally, another upstream regulator, NR5A2, identified in our datasets, is involved in lipid metabolism. NR5A2 plays critical roles in embryonic development, cholesterol and bile acid homeostasis, and cell proliferation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). NR5A2 has also been shown to indirectly regulate the immune system and associated inflammatory processes via the synthesis of immunoregulatory glucocorticoids in the intestinal crypts (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Tissue-specific deletion or inhibition of NR5A2 and associated intestinal glucocorticoid synthesis consequently results in increased susceptibility to the development of intestinal inflammatory disorders (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Based on our analysis, the upstream regulators MEF2C, KDM5A, and NR5A2, along with the downregulation of their target genes (Ccl9, COL10A1, CXCL2, MMP8, Ccl7, IL1B, IL6, SOD2, and others), may represent an additional mechanism by which Fer-1 exerts its therapeutic effects in AD. This mechanism likely involves the modulation of immune responses, oxidative stress, apoptosis, and lipid metabolism, as well as the prevention of inappropriate phenotypic alterations in SMCs and the enhancement of endothelial barrier function.\u003c/p\u003e \u003cp\u003eIn addition to epigenetic regulation, miRNAs were also implicated in the protective effects of Fer-1. MiR-361-5p, previously associated with acute coronary syndrome and endothelial cell function (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The expression of miR-361-5p was significantly decreased in ox-LDL injured vascular SMCs, while lncRNA MEG3-derived miR-361-5p regulate vascular SMC proliferation and apoptosis by targeting ABCA1(\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In this study, miR-361-5p was predicted to be upregulated following Fer-1 treatment. It was identified as an upstream regulator of genes such as HMOX1, ATP1B4, CACNG1, G6PC1, and HOMER1, which are involved in modulating oxidative stress, inflammation, and ion homeostasis. Previous research has established a strong association between HMOX1 and ferroptosis (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) ), with excessive HMOX1 expression potentially triggering ferroptosis. Conversely, miR-3151-5p expression was found to be downregulated in Fer-1-treated mice. This miRNA is predicted to target genes related to cell death (e.g., CASP3, CASP9, FAS), ion homeostasis (e.g., CALB1), and inflammation (e.g., IL6, IL1B, NLRP3). Notably, the downregulation of IL6 and IL1B, key inflammatory mediators, observed in Fer-1-treated mice corresponds with the reduction in inflammation, further supporting the therapeutic effects of Fer-1.\u003c/p\u003e \u003cp\u003eThe plasticity of SMCs is crucial for vascular compliance and ECM regulation, particularly in aortic disease pathogenesis. PAI-1 inhibits cofilin, a key cytoskeletal regulator, thereby influencing SMC stiffness and F-actin content (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Consistent with our findings, Fer-1 treatment regulated cytoskeleton polymerization by reducing F-actin level (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). ILK an intracellular serine/threonine kinase, plays a critical role in cell-matrix interactions and signal transduction (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). It induces AKT phosphorylation and p21 degradation, which are associated with SMC migration and proliferation (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Furthermore, ILK activates MMP-9 promoter, and consequently MMP-9 expression through the GSK-3β/AP-1 pathway (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) and contributes to Ang-II-induced renal inflammation (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). IL-17 plays a critical role in acute inflammation and is reported to participate in AD pathogenesis (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) by interfering with TGF-β signal and altering ECM metabolism (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Additionally, Fer-1 treatment downregulated pro-inflammatory M1 macrophage pathways while upregulating anti-inflammatory M2 macrophage pathways (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Findings form our datasets according enrichment analysis on canonical pathways of IPA suggest that Fer-1 preserves SMC differentiation and attenuates ECM remodeling through its anti-inflammatory and cytoskeletal regulatory effects.\u003c/p\u003e \u003cp\u003eGene interaction network analysis further revealed key molecules such as CXCR3, ACACA, and BPGM, which play roles in inflammation, lipid metabolism, and vascular wall integrity. CXCR3 regulates immune cell recruitment and inflammatory signaling, processes exacerbating aortic wall damage. ACACA, a key enzyme in fatty acid biosynthesis, influences lipid homeostasis, while BPGM reflects metabolic adaptation under oxidative stress. These interactions indicate that Fer-1 not only inhibits ferroptosis but also modulates broader metabolic and inflammatory pathways to support vascular health.\u003c/p\u003e \u003cp\u003eOther insightful pathway in this study identified 922 DEGs associated with ferroptosis inhibition, including pathways related to phagocytes response, occlusion of blood vessel, and ROS synthesis to against AD (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e-\u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e). While this research provides compelling evidence for the role of ferroptosis in AD and the protective effects of Fer-1, certain limitations remain. Functional validation of identified DEGs and their roles in ferroptosis and AD pathogenesis is necessary. Additionally, transgenic animal studies could clarify the contributions of targets such as MEF2C and KDM5A. The acute effects of Fer-1 were the primary focus; future studies should investigate its long-term efficacy and potential side effects. Dose-response studies are also needed to optimize therapeutic outcomes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis research elucidates the mechanism of ferroptosis in AD pathogenesis and establishes Fer-1 as a promising therapeutic intervention. By attenuating ferroptosis-induced lipid peroxidation, oxidative stress, and inflammation, Fer-1 effectively preserves aortic wall integrity and potentially reduces AD incidence and mortality. The findings highlight the therapeutic potential of ferroptosis-targeted interventions and provide a foundation for future translational research.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAortic Dissection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFer-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFerrostatin-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSMC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSmooth Muscle Cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular Matrix\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBAPN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eβ-aminopropionitrile\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAng-II\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAngiotensin II\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSPF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSpecific Pathogen-free\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIngenuity Pathway Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrincipal Component Analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially Expressed Genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFold Change\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Wan Fang Hospital, Taipei Medical University (112-wf-eva-23) and the National Science and Technology Council, Taiwan (NSTC 112-2314-B-038 -122 -MY3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChun-Che Shih*: writing \u0026ndash; original draft, writing \u0026ndash; review and editing, data curation. Chi-Yu Chen *: writing \u0026ndash; original draft, writing \u0026ndash; review and editing, formal analysis, methodology. Chih-Pin Chuu: formal analysis, writing \u0026ndash; review and editing, visualization. Chun-Yang, Huang: formal analysis, writing \u0026ndash; review and editing. Chia-Jung Lu: methodology, writing \u0026ndash; review and editing. Hsin-Ying Lu: conceptualization, writing \u0026ndash; review and editing, funding acquisition, project administration.\u003c/p\u003e\n\u003cp\u003e*These authors contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data presented in the present study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted with the approval of Laboratory Animal Center of Taipei Medical University (Protocol number: No: LAC-2022-0469).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNienaber CA, Clough RE, Sakalihasan N et al (2016) Aortic dissection. 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Arterioscler Thromb Vasc Biol 40:189\u0026ndash;205\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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, Ferroptosis, Inflammation, Transcriptomics","lastPublishedDoi":"10.21203/rs.3.rs-6061217/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6061217/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAortic dissection (AD) is a life-threatening vascular condition with limited pharmacological options. This study investigated Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, in a BAPN/Ang-II-induced mouse model of AD, revealing significant therapeutic potential. Fer-1 significantly reduced AD incidence and mortality by preserving aortic wall integrity. RNA sequencing identified 922 differentially expressed genes, with 416 upregulated and 506 downregulated. Bioinformatics analysis revealed that Fer-1 modulates key regulators, such a\u003cb\u003es\u003c/b\u003e MEF2C and KDM5A, impacting immune responses, oxidative stress, apoptosis, and lipid metabolism. Additionally, Fer-1 alters miRNA expression, with the upregulation of miR-361-5p and downregulation of miR-3151-5p, targeting pathways involved in inflammation, oxidative stress, and smooth muscle cell (SMC) phenotypic stability. Functional pathway analysis highlighted the inhibition of actin cytoskeleton, ILK, and IL-17 signaling, essential for SMC differentiation and extracellular matrix remodeling. Gene interaction network analysis identified 21 central molecules, including CXCR3, ACACA, and BPGM, associated with lipid metabolism, inflammation, and vascular remodeling. These findings demonstrate Fer-1's potential to mitigate ferroptosis and modulate inflammation, offering a comprehensive protective mechanism against AD and potentially other ferroptosis-driven vascular diseases.\u003c/p\u003e","manuscriptTitle":"Transcriptome Insights into Protective Mechanisms of Ferroptosis Inhibition in Aortic Dissection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-21 15:07:03","doi":"10.21203/rs.3.rs-6061217/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":"f600b12f-87bb-469a-832d-d003f35ef655","owner":[],"postedDate":"February 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-23T18:53:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-21 15:07:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6061217","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6061217","identity":"rs-6061217","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}