Chromobox 3 Assembles a Novel Epigenetic Complex Contributing to Cystathionine γ-lyase–mediated Protection Against Aortic Aneurysm/dissection

Chromobox 3 Assembles a Novel Epigenetic Complex Contributing to Cystathionine γ-lyase–mediated Protection Against Aortic Aneurysm/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 Article Chromobox 3 Assembles a Novel Epigenetic Complex Contributing to Cystathionine γ-lyase–mediated Protection Against Aortic Aneurysm/dissection Bin Geng, Ying Zhao, Changting Cui, Huimin Gao, Yaping Niu, Ling Cheng, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7423522/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Hydrogen sulfide (H₂S), generated by cystathionine γ-lyase (CSE), exerts protective effects against aortic aneurysm/dissection (AAD). Proteoglycans, major extracellular matrix (ECM) components synthesized by vascular smooth muscle cells (VSMCs), preserve aortic wall integrity but also contribute to AAD pathogenesis. The mechanisms linking VSMC-derived CSE/H₂S to proteoglycan regulation in AAA remain undefined. Here, we identified reduced CSE expression in VSMCs (α-SMA⁺) from human AAD tissues and murine models. VSMC-specific CSE deletion (CSE SMCKO ) exacerbated AngII-induced AAD, with increased ADAMTS4 expression and versican degradation. Mechanistically, CSE loss suppressed CBX3, releasing Adamts4 transcriptional repression. Conversely, CBX3 overexpression ameliorated AAD in CSE SMCKO mice. CBX3 formed an epigenetic complex with SUV39H1, KDM2A, HDAC1, and RING1, regulating H3K9/H3K4 methylation/acetylation, thereby modulating ECM remodeling, apoptosis, and inflammation. Therapeutically, AAV-mediated CSE or CBX3 delivery via extravascular carrier reduced AAD incidence and progression. Thus, VSMC-derived CSE/H₂S–CBX3 signaling restrains AAD through epigenetic regulation of the ADAMTS4–versican axis. Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases Health sciences/Diseases/Cardiovascular diseases Abdominal aortic aneurysm Cystathionine γ lyase Hydrogen sulfide Vascular smooth muscle cells A disintegrin-like and metalloproteinase with thrombospondin motifs 4 Chromobox 3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Aortic aneurysm/dissection (AAD) is a chronic aortic disorder characterized by progressive weakening and dilation of the vessel wall, frequently culminating in fatal rupture and accounting for an estimated 150,000–200,000 deaths annually worldwide 1 , 2 . Hallmark pathological features include vascular smooth muscle cell (VSMC) loss, extracellular matrix (ECM) degradation (particularly elastin and collagen), proteoglycan accumulation, and localized inflammation 3 , 4 . VSMCs are central to maintaining aortic integrity through ECM synthesis, regulation of cell death, and modulation of inflammatory responses 3 . The aortic ECM is a dynamic network composed of fibrous proteins (collagen, elastin, fibrillins) and proteoglycans such as versican and aggrecan 4 , 5 . Pathological ECM remodeling in AAD is driven by proteases, notably matrix metalloproteinases (MMPs), which degrade structural proteins, and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteoglycanases 6 . Versican, a large chondroitin sulfate proteoglycan predominantly expressed in VSMCs 7 , plays a critical role in vascular homeostasis 8 . Versican accumulation is consistently observed in human and experimental AAD 9 , 10 . Within the ADAMTS family, six members (ADAMTS1, 4, 5, 9, 15, 20) possess proteoglycanase activity 11 . ADAMTS5 exhibits the highest activity against versican, followed by ADAMTS4 and ADAMTS1 12, 13 . Notably, ADAMTS4—upregulated in both clinical and experimental AAD—cleaves versican at the Glu441-Ala442 site 14 , 15 , whereas ADAMTS5 is paradoxically downregulated 16 . Genetic studies confirm this functional divergence: ADAMTS4 deficiency is protective against aneurysm, while loss of ADAMTS1 or ADAMTS5 accelerates disease 11 , 17 , 18 . Together, these findings underscore the importance of versicanolysis in ECM remodeling and AAD pathogenesis. Hydrogen sulfide (H₂S), an endogenous gasotransmitter synthesized mainly by cystathionine γ-lyase (CSE), exerts vasoprotective effects via protein sulfhydration 19 . Clinical and experimental studies consistently report reduced CSE expression and H₂S bioavailability in AAD 20 , 21 . Endothelial-specific CSE deficiency exacerbates dissection through enhanced endoplasmic reticulum stress, a process reversed by H₂S-mediated sulfhydration of protein disulfide isomerase 21 . H₂S also inactivates MMP-2 and MMP-9 by sulfhydrating their cysteine-switch motifs 22 . While VSMCs produce MMPs, major sources of MMP-2 and MMP-9 in dissection are fibroblasts/leukocytes and macrophages, respectively 23 . Importantly, VSMCs are the predominant producers of vascular H₂S 24 . Thus, although H₂S-mediated MMP inactivation may partly account for CSE/H₂S vasoprotection, the broader regulatory mechanisms, particularly concerning ADAMTS proteases and proteoglycan turnover, remain poorly defined. Here, we show that VSMC-specific CSE deletion aggravates AAD through epigenetic dysregulation of the ADAMTS4–versican axis. Mechanistically, CSE deficiency suppresses chromobox 3 (CBX3), disrupting histone H3K4/H3K9 methylation and acetylation, thereby derepressing Adamts4 transcription. We further identify CBX3 as a scaffold for a previously unrecognized histone-modifying complex integrating methylation and acetylation pathways. Finally, local delivery of CSE or CBX3 via absorbable extravascular carrier mitigates AAD progression, highlighting a promising translational strategy. Methods The data, research methods, and study materials utilized in this study are available from the corresponding authors on reasonable request. Detailed Expanded Methods section are provided in the Supplemental Material. Human Aortic Samples The human aortic tissues were collected in compliance with the Decleration of Helsinki. The AAD aortic samples were collected from patients who suffered aneurysmectomy and vascular transplantation. The non-AAD samples were from the Biospeciman Bank of the First Affiliated Hospital of Harbin Medical University. The baseline characteristics of the patients are presented in Supplementary Table 1. The study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (2025141). Animal Study and AAD Models Animal experiments were approved by Institute of Animal Care and Use Committee of Fuwai Hospital, Chinese Academy of Medical Sciences (FW-2020-0059), and and performed in accordance with the animal experiment guidline. CSE SMCKO knockout mice generated by CSE LOXP (containing loxP sites flanking exons 2 and 3) hybridzation with Tagln-Cre mice as our previous study 24 . ApoE knockout mice (ApoE -/- ) and C57BL/6J mice were purchased by Charles River Laboratories (Beijing, China). AngII-induced AAD model was performed as previously described 25 . Briefly, 8- to 10-week-old male ApoE -/- mice were subcutaneously implanted with minipumps (Alzet, Model 2004) to infuse AngII (1000ng/kg/min) for 4 weeks while being maintained on a Paigen diet to induce AAD. The PCSK9/AngII-induced AAD model was established following established protocols 26 . Briefly, 8- to 10-week-old mice (CSE LOXP , CSE SMCKO ) received intravenous injection of recombinant adeno-associated virus (rAAV8-HCRApoE/hAAT-D377Y-mPCSK9; 5×10¹¹ vector genome copies in 150 µl sterile saline) while being maintained on a Paigen diet to induce hyperlipidemia. One week post-AAV administration, AngII (1,000 ng/kg/min) was continuously delivered via subcutaneous osmotic minipump for 4 weeks to promote AAD development. The β-aminopropionitrile (BAPN)/AngII-induced AAD model was established as previously described 27 . Briefly, 8- to 10-week-old mice received concurrent administration of AngII (1,000 ng/kg/min) via osmotic minipump and BAPN (150 mg/kg/day) in drinking water for 4 weeks to induce aortic aneurysm formation. Statistical Analysis Data are presented as mean with standard error of the mean (SEM). Differences between two groups were evaluated with unpaired 2-tailed T-test (normal distribution), Mann-Whitney test (non-normal distribution), or Fisher's exact test (categorical data). For more groups, data were compared by one-way ANOVA followed by post-hoc analysis. Comparisons including two factors were performed by two-way ANOVA, with repeated measures on the same animals or cells analyzed via two-way mixed-effects ANOVA. All statistical analysis involved using GraphPad Prism v10.1.2. P < 0.05 was considered statistically significant. Results Down-regulated VSMC CSE expression in AAD To examine the alterations of CSE expression in VSMCs within AAD, we performed immunofluorescence staining. Compared with healthy aortic tissues, CSE staining (green) in α-SMA–positive VSMCs (red) was markedly reduced in AAD patients (Fig. 1 A). This observation is consistent with prior reports demonstrating decreased CSE expression in diseased aortic tissues, further supported by Western blot analyses 20 , 21 . In the aorta of ApoE −/− mice, CSE expression was detectable in both endothelial cells and VSMCs, but significantly decreased in VSMCs within the AngII-induced AAD model (Fig. 1 B, Supplementary Fig. 1). In vitro, AngII treatment notably suppressed both CSE mRNA and protein expression in human and mouse aortic smooth muscle cells (Fig. 1 C–F). Collectively, evidence from human samples, animal models, and cultured cells demonstrates that VSMC-derived CSE expression is downregulated during AAD development. VSMC-Specific Deletion of CSE Promotes AAD Progression To determine the functional role of CSE in AAD pathogenesis, we assessed phenotypic changes in two AngII-induced AAD mouse models. In the first model, AAD was established by overexpressing PCSK9 in the liver using AAV8-D377Y-mPCSK9 combined with a Paigen diet to induce hypercholesterolemia, followed by AngII infusion for four weeks. VSMC-specific CSE deletion (validated in Supplementary Fig. 2A, B) significantly increased AAD incidence (95.4% vs. 48%; Fig. 2 A), mortality (22% vs. 7%; Fig. 2 B), abdominal aortic diameter (1.96 mm vs. 1.42 mm; Fig. 2 C), and elastin degradation (Fig. 2 D) compared with controls. To further confirm these results, we employed a second AAD model combining BAPN administration with AngII infusion. Consistent with findings from the AAV-PCSK9 + AngII model, CSE deletion in VSMCs markedly elevated AAD incidence (95.6% vs. 34.4%; Fig. 2 E), mortality (60% vs. 16%; Fig. 2 F), aortic diameter (1.28 mm vs. 0.92 mm; Fig. 2 G), and elastin degradation (Fig. 2 H). Moreover, CSE-deficient VSMCs displayed enhanced apoptosis (Supplementary Fig. 2C) and increased expression of MMP2 and MMP9 (Supplementary Fig. 2D). These results strongly indicate that CSE deficiency in VSMCs accelerates AAD initiation and progression. CSE Deficiency in VSMCs Induces ADAMTS4 Accumulation and Versican Degradation ECM remodeling is a hallmark of AAD pathogenesis, driven by MMP-mediated degradation of collagen and elastin and ADAMTS-mediated degradation of proteoglycans. To elucidate the role of CSE in ECM regulation, we performed RNA sequencing of AngII-stimulated VSMCs. Transcriptomic analysis (GEO: GSE290627) revealed significant alterations in MMP and ADAMTS family members: Mmp9 , Mmp10 , Mmp13 , Adamts3 , and Adamts4 were upregulated, whereas Adamts9 and Adamts10 were downregulated (Fig. 3 A, Supplementary Fig. 3A). qRT-PCR validated these results, except for Adamts10 (Fig. 3 B, Supplementary Fig. 3B), in CSE-deficient VSMCs following AngII exposure. Protein analysis confirmed that CSE knockout augmented ADAMTS4 expression and enhanced versican degradation in VSMCs upon AngII (1 µM) stimulation for 24 hours (Fig. 3 C). Conversely, CSE overexpression suppressed ADAMTS4 expression and reduced versican degradation (Fig. 3 D). Pathological staining further demonstrated elevated ADAMTS4 levels in α-SMA–positive aortic VSMCs and increased accumulation of the versican cleavage fragment V1 in CSE SMCKO compared with CSE LOXP mice across both AAD models (Fig. 3 E). Importantly, increased ADAMTS4 expression was also detected in VSMCs from human AAD tissues (Fig. 3 F) and in the AngII-induced ApoE −/− mouse model (Fig. 3 G), coinciding with pronounced versican degradation. These findings, consistent with previous studies 14 , 15 , underscore a critical role of the ADAMTS4–versican axis in mediating CSE-regulated ECM remodeling during AAD pathogenesis. CBX3 Negatively Regulates ADAMTS4 Expression Polycomb repressive complexes (PRCs), comprising PRC1 and PRC2, act as key epigenetic regulators by modifying histones to suppress gene transcription. To identify PRC components involved in Adamts4 regulation, we analyzed downregulated genes from RNA-seq data and validated them by qRT-PCR. Both Cbx3 and Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit ( Ezh2 ) were reduced following CSE deletion (Fig. 4 A, Supplementary Fig. 4A). Functional studies revealed that siRNA-mediated knockdown of Ezh2 decreased Adamts4 transcription, whereas Cbx3 knockdown markedly increased Adamts4 expression (Supplementary Fig. 4B, C), indicating a repressive role of CBX3. Western blot analysis confirmed reduced CBX3 protein levels after CSE deletion under AngII or ox-LDL stimulation (Fig. 4 B). Immunofluorescence demonstrated that CBX3 predominantly localized in the nucleus but was diminished in CSE-deficient VSMCs (Fig. 4 C), whereas CSE overexpression enhanced CBX3 expression (Fig. 4 D). In vivo, CBX3 expression was significantly reduced in VSMCs of CSE SMCKO mice compared with controls in two AAD models (Fig. 4 E). Similar downregulation was observed in VSMCs from AAA patients and ApoE −/− mice (Fig. 4 F). To define its functional role, primary VSMCs from CSE-knockout mice were studied. CBX3 knockdown further elevated ADAMTS4 accumulation and versican cleavage (Fig. 4 G), while CBX3 overexpression suppressed both (Fig. 4 H). These findings establish CBX3 as a nuclear mediator linking endogenous CSE/H₂S signaling to ADAMTS4 regulation. CBX3 Overexpression Attenuates CSE Deficiency–Induced AAD To determine whether restoring CBX3 could mitigate AAD progression driven by CSE deficiency, we developed a localized gene delivery system. A collagen-based absorbable “extravascular stent-like carrier” was implanted around the superior renal artery, adsorbing AAV9-sm22α-Cbx3 to achieve targeted CBX3 overexpression; AAV9-sm22α-GFP served as control (Supplementary Fig. 5). One week later, mice were subjected to BAPN + AngII to induce AAD. Among survivors (14/group), CBX3 overexpression significantly reduced AAD incidence (4/14 vs. 12/14; Fig. 5 A, B), aortic dilation (Fig. 5 C), elastin degradation, and collagen deposition (Fig. 5 D). Moreover, pathological hallmarks of AAD progression—including versican cleavage (Fig. 5 E), ADAMTS4 accumulation (Fig. 5 F), VSMC apoptosis (TUNEL staining, Fig. 5 G), macrophage infiltration (CD68 positive cells), and elevated MMP2/9 (Fig. 5 H)—were all attenuated by CBX3 overexpression. Collectively, these data demonstrate that CBX3 restoration rescues the exacerbated AAD phenotype in CSE-deficient mice, primarily via the CBX3–ADAMTS4–versican signaling axis. Histone Modifications Mediate CBX3’s Response to CSE Deletion CBX3 is tightly linked to histone modifications across various cell types 28 , 29 . To test whether these modifications contribute to CBX3-mediated regulation, we assessed histone marks under CSE deletion. AngII or ox-LDL stimulation in CSE-deficient VSMCs reduced H3K9me3, H3K9ac, H3K4me3, and H3K4ac, while H3K27me3 remained unchanged (Fig. 6 A). Conversely, CSE overexpression enhanced H3K4/9 methylation and acetylation (Fig. 6 B). In vivo, CSE SMCKO mice exhibited reduced H3K9me3/ac and H3K4me3/ac levels in the aorta across two AngII-induced AAA models (Fig. 6 C, D). Similar reductions were confirmed in human AAA tissues (Supplementary Fig. 6A) and ApoE −/− mice (Supplementary Fig. 6B), suggesting that impaired H3K4/9 methylation/acetylation represents a common epigenetic mechanism in AAD. CBX3 was further implicated in this process: knockdown in CSE-deficient VSMCs aggravated loss of H3K4/9 methylation/acetylation (Fig. 6 E), whereas CBX3 overexpression restored these modifications under AngII stimulation (Fig. 6 F). These results indicate that CBX3 is a key mediator of CSE-induced histone modifications. CSE–CBX3–Histone Modification Axis in AAD To delineate the molecular mechanisms, we performed ChIP-seq and RNA-seq integration. CSE deletion increased H3K9me3 enrichment near transcription start sites (TSS ± 3 kb; Fig. 7 A), affecting 355 genes (92 upregulated, 263 downregulated). GO and KEGG analyses revealed enrichment in ECM regulation, apoptosis, VSMC function, and inflammation pathways (Fig. 7 A, Supplementary Fig. 7). Conversely, CSE deletion reduced H3K9ac and H3K4me3 enrichment (Fig. 7 B, C). IGV profiles highlighted decreased histone modifications in ECM-related ( Adamts4 , Mmp9 , Col1a2 ), inflammation-related ( Nlrp3 ), and apoptosis-related ( Bcl2 ) genes (Fig. 7 D, E). qRT-PCR validated upregulation of Adamts4 (Fig. 4 B), Mmp9 (Supplementary Fig. 3B), and Nlrp3 , alongside downregulation of Col1a2 and Bcl2 in CSE-deficient VSMCs (Fig. 7 F). These findings collectively demonstrate that the CSE–CBX3 axis orchestrates histone modifications at H3K4/9, thereby regulating ECM remodeling, apoptosis, and inflammation pathways central to AAD development. CBX3 as a Component of a Novel Epigenetic Regulatory Complex CBX3, a heterochromatin protein 1 (HP1)–interacting factor, is known to function as an H3K9me3 methyl-reader; however, its role in modulating other histone marks such as H3K9ac, H3K4me3, and H3K4ac remains unclear. To identify CBX3-binding partners, we performed immunoprecipitation (IP) followed by proteomic analysis. SDS-PAGE with silver staining (Supplementary Fig. 8A) revealed distinct protein bands, which were analyzed by mass spectrometry, identifying 405 potential CBX3 interactors (Supplementary Table 3). GO and KEGG analyses showed significant enrichment in histone binding and modification pathways (Supplementary Fig. 8B). For validation, plasmids encoding Flag-tagged CBX3, HA-tagged KDM2A, Myc-tagged HDAC1, His-tagged RING1, and Strep-tagged SUV39H1 were co-transfected in random pairs into HEK293T cells. Co-immunoprecipitation confirmed CBX3 interactions with RING1, SUV39H1, KDM2A, and HDAC1 (Fig. 8 A). These interactions were further validated in primary mouse aortic VSMCs (Fig. 8 B). Notably, CSE deletion disrupted CBX3 interactions with KDM2A and SUV39H1, while strengthening its association with HDAC1 (Fig. 8 C). Collectively, these findings indicate that CBX3 forms a dynamic epigenetic regulatory complex with SUV39H1, KDM2A, HDAC1, and RING1, responsive to changes in the CSE/H₂S system, thereby contributing to AAD pathogenesis. Local Overexpression of CSE or CBX3 Attenuates AAD Development To assess the therapeutic potential of CSE or CBX3 restoration, we implanted a collagen sponge–based absorbable extravascular stent around the abdominal aorta (between the diaphragm and renal artery) loaded with AAV9-sm22α-CSE or AAV9-sm22α-CBX3. ApoE −/− mice underwent AngII infusion one week after implantation. Four weeks later, both CSE and CBX3 overexpression markedly reduced AAD incidence (Fig. 9 A), aortic diameter (Fig. 9 B), elastin degradation, and collagen deposition (Fig. 9 C). Additionally, apoptotic cell counts (TUNEL, Supplementary Fig. 9A), macrophage infiltration (CD68 staining), and MMP2/MMP9 expression (Supplementary Fig. 9B) were significantly decreased in both treatment groups. Consistent with these phenotypic changes, CSE or CBX3 overexpression also reduced ADAMTS4 accumulation and versican cleavage in the aortic media (Fig. 9 D, E). Interestingly, CSE overexpression partially restored CBX3 expression to levels comparable with direct CBX3 overexpression (Fig. 9 D), whereas CBX3 overexpression did not affect CSE expression (Supplementary Fig. 9A), confirming CBX3 as a downstream effector of CSE in vivo. Together, these findings demonstrate that targeted, VSMC-specific overexpression of CSE or CBX3 via AAV-based delivery using an absorbable extravascular carrier effectively suppresses AAD progression, highlighting a novel therapeutic strategy based on the CSE–CBX3–ADAMTS4 axis. Discussion VSMCs are the main producers of CSE and H₂S in arterial tissues, regulating VSMC functions such as contraction, proliferation, migration, apoptosis and senescence 19 , 24 , 30 . H₂S influences key proteins via sulfhydration, inhibiting MMP2/MMP9 activity 22 and reducing endoplasmic reticulum stress 21 , 31 , thereby modulating AAD progression. However, direct evidence linking VSMC-specific CSE/H₂S signaling to AAD remains limited. In this study, we observed reduced CSE expression in VSMCs from AAD patients and murine models. Using a conditional knockout mouse model, we found that CSE deletion in VSMCs worsened AAD in two AngII-induced models. Mechanistically, CSE loss disrupted the CBX3 epigenetic complex, altering histone modifications and promoting Adamts4 transcription, and dysregulation of Mmp9 , Col1a2 , Nlrp3 , and Bcl2 . These changes exacerbated versican cleavage, collagen/elastin degradation, inflammation, and apoptosis, accelerating AAD development (Supplementary Fig. 9). Previous studies have shown reduced aortic CSE expression in AAA and aortic dissection patients, as well as in the aortic endothelium of AngII-induced ApoE −/− mice 20, 21 . Our study further confirms CSE downregulation in VSMCs of AAD patients and an AngII-induced mouse model using immunofluorescence staining. Risk factors for AAD, such as aging (including cell senescence and senility), male gender, hypertension, hypercholesterolemia, and diabetes (high glucose) 32 , Notably, several studies have reported that high glucose levels, hypercholesterolemia, are linked to decreased VSMC CSE expression 17 , 23 , 27 . AngII, a key AAD inducer, directly suppresses CSE in VSMCs and endothelial cells 21 , 30 , 33 , via transcriptional repression by the ZEB2-HDAC1-NuRD complex and post-translational including HDAC6-mediated acetylation at K73 and ubiquitination at K48, leading to CSE degradation 21 , 33 , 34 , 35 . These findings highlight how risk factors accelerate CSE reduction in aortic VSMCs, contributing to AAD pathogenesis and progression. The histopathological hallmark of ECM remodeling in AAD is characterized by the degradation and disorganization of elastic and collagen fibers, along with proteoglycan accumulation 6 . Among the major large proteoglycans, versican and aggrecan play crucial roles in maintaining the reversible compressive structure of the aortic wall, regulating VSMC homeostasis, and are notably upregulated in thoracic aortic aneurysm and dissection (TAAD) 9 , 36 . The ADAMTS family members (ADAMTS1, ADAMTS4, and ADAMTS5) exhibit proteolytic activity toward aggrecan and versican 7 , with their protein expression significantly elevated in TAAD patients 6 . Genetic studies in mice reveal divergent roles for these proteases: Adamts1 heterozygosity exacerbates high-fat diet plus AngII-induced aortic events but attenuates BAPN plus AngII-induced pathology 17 , 37 . Deletion of the Adamts5 catalytic domain enhances AngII-induced ascending aortic dilation 18 . Global Adamts4 knockout mitigates AngII-induced AAD formation, versican degradation, elastic fiber destruction, macrophage infiltration, and VSMC apoptosis 15 . In our current study, smooth muscle cell-specific CSE knockout mice exhibited exacerbated AngII-induced AAD incidence and progression, correlating with ADAMTS4 accumulation and heightened versican degradation. Consistent with prior findings on ADAMTS4-versican dysregulation, we observed concomitant increases in elastic fiber degradation, macrophage infiltration, and VSMC apoptosis, mirroring the phenotypic consequences of Adamts4 deletion 15 . RNA-seq analysis further confirmed that Adamts1 and Adamts5 expression remained unchanged in CSE-deficient VSMCs compared to wild-type controls. These findings collectively implicate ADAMTS4-versican axis dysregulation as a key mechanism underlying ECM remodeling, inflammatory responses, and cell death in AAD pathogenesis, mediated by VSMC CSE/H₂S signaling. Importantly, human AAD samples corroborated these results, demonstrating elevated ADAMTS4 expression and versican degradation—phenotypes also observed in TAAD 14 , 15 , and traditional murine AAD models. Therefore, ADAMTS4 upregulation represents a conserved pathophysiological mechanism in AAD, suggesting that targeted inhibition of this protease may hold therapeutic potential for preventing or treating AAD. ADAMTS4 transcription is bidirectionally regulated by SP1/AP-2α (activators) and nuclear factor I (NFI)/histone H4 deacetylation (repressors) 38 , 39 . While CSE/H 2 S inhibits SP1 via sulfhydration-mediated suppression of Jumonji domain-containing protein 3 and MMP2 22, 40 , its effects on AP-2α/NFI/H4 remain unknown. Here, we identified CBX3 as a novel negative regulator of Adamts4 transcription. CBX3 levels were significantly reduced in both CSE-deficient VSMCs/aortic tissues and human/murine AAA specimens. Functional studies demonstrated that localized CBX3 overexpression in CSE-deficient VSMCs attenuated AAD progression, concomitant with decreased ADAMTS4 expression and versican cleavage. These findings establish CBX3 as a previously unidentified transcriptional repressor in the ADAMTS4 regulatory network, providing new mechanistic insights into AAD pathogenesis. The CBX family proteins are canonical components of PRC1, which is a well-characterized transcriptional repressor primarily mediating gene silencing through H2AK119 ubiquitination 37 . CBX3 exhibits dual functions as both a PRC1 component and an H3K9me3 reader through its interaction with HP1 complexes and SUV39H1/2 methyltransferases 41 . It regulates diverse processes including VSMC proliferation and migration (via Notch3 signaling) 42 , interfering artery development (via smooth muscle cells differentiation) 43 , and H4K20me3-dependent cardiac growth 28 . Recent studies show CBX3 recruits EP300 to promote histone lactylation 44 . In this study, we identify CBX3 as a specific regulator of H3K9me3/ac and H3K4me3/ac, but not H3K27me3. Proteomic and Co-IP analyses reveal CBX3 forms a novel epigenetic complex with SUV39H1, KDM2A, HDAC1, and RING1. These interactions suggest the formation of a novel histone-modifying complex centered around CBX3. ChIP-seq also demonstrates CSE-induced CBX3 downregulation alters these marks at loci involved in ECM remodeling, apoptosis, and inflammation - key pathways in AAD pathogenesis. We propose CBX3 serves as a scaffold coordinating methylase/demethylase (SUV39H1/KDM2A), acetyltransferase/deacetylase (EP300/HDAC1), and ubiquitin ligase (RING1) activities, forming a transcriptional repressor module that drives AAD development through integrated histone modification control. Open surgery (OS) and endovascular aneurysm repair (EVAR) are the primary therapeutic strategies for AAD. Short-term outcomes favor EVAR due to its advantages, including shorter operation times, reduced intraoperative blood loss, decreased need for blood transfusions, and lower rates of postoperative mechanical ventilation 45 , 46 , 47 . However, long-term mortality rates do not significantly differ between OS and EVAR, likely because EVAR requires more frequent secondary interventions—often due to graft-related complications such as endoleaks 45 . To address these limitations, we developed a novel "extravascular stent-like carrier" composed of a collagen sponge, which offers several key benefits: (1) prevention of aneurysm rupture, (2) biodegradability, and (3) the capacity to deliver therapeutic agents (e.g., viruses or drugs) for localized treatment. In our approach, the carrier was implanted around the abdominal aorta between the diaphragm and renal artery—the most susceptible site for aneurysm formation in an AngII-induced AAD mouse model. This placement effectively reduced aneurysm rupture. Additionally, we loaded the carrier with AAV9 vectors to overexpress CSE or CBX3 in VSMCs, which significantly attenuated AngII-induced AAD incidence and progression. Collectively, our findings suggest that implantation of an extravascular stent combined with localized gene therapy may represent a promising alternative therapeutic strategy for AAD. Limitations of the current study include the following: 1) Lack of VSMC-specific CBX3 inducible knockout mice. These mice would have been valuable to confirm whether the protective effects of H 2 S donors on AAD are mediated through CBX3. 2) The study did not explore how CSE/H 2 S signaling regulates CBX3 expression or its regulatory model of the novel epigenetic complex. 3) Only the AngII-induced AAD model was used; other established models, such as elastase-induced or CaCl2-induced aortic aneurysm, were not examined. 4) Key translational questions of extravascular stent challenges in large animals remain unresolved, including material optimization, biomechanical parameters, and therapeutic efficacy in preclinical large-animal AAD models. In summary, this study elucidates the critical role of VSMC-derived CSE/H 2 S in the pathogenesis of AAD and uncovers a novel epigenetic regulatory mechanism governing ECM remodeling, cellular apoptosis, and inflammatory responses in AAD progression. Furthermore, we identified a previously unrecognized epigenetic complex composed of CBX3, SUV39H1, KDM2A, HDAC1, RING1, and HAT 44 , which collectively modulate the methylation and acetylation of histone H3K9 and H3K4 to regulate gene expression. From a translational perspective, we developed an innovative therapeutic approach combining an extravascular stent with localized transgene delivery, offering a potential strategy for AAD intervention. This dual-component system not only provides structural support to attenuate aneurysm progression but also enables targeted modulation of key molecular pathways implicated in AAD pathogenesis. Abbreviations AAA Abdominal Aortic Aneurysm CSE Cystathionine γ lyase H 2 S Hydrogen Sulfide VSMCs Vascular Smooth Muscle Cells ECM Extracellular Matrix ADAMTS A Disintegrin-like and Metalloproteinase with Thrombospondin Motifs CBX3 Chromobox 3 AAV Adeno-Associated Virus PCSK9 Proprotein Convertase Subtilisin/Kexin type 9 ApoE Apolipoprotein E PRC Polycomb Repressive Complex BAPN β-aminopropionitrile ChIP-seq Chromatin Immunoprecipitation sequencing TSS Transcription start site GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes HP1 Heterochromatin Protein 1 MMP Matrix Metalloproteinases NLRP3 NOD-like receptor protein family pyrin domain containing 3 AAD Aortic aneurysm and dissection TAAD Thoracic aortic aneurysm and dissection HAT Histone acetyltransferase OS Open surgery EVAR Endovascular aneurysm repair H3K4/9me3 Histone 3 lysine 4/9 trimethylation H3K4/9ac Histone 3 lysine 4/9 acetylation H3K27me3 Histone 3 lysine 27 trimethylation α-SMA α-smooth muscle actin EZH2 Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit ox-LDL oxidized Low-Density Lipoprotein HDAC1 Histone Deacetylase 1 KDM2A Lysine Demethylase 2A SUV39H1 SUV39H1 Histone Lysine Methyltransferase RING1 Ring Finger Protein 1 BCL2 BCL2 Apoptosis Regulator COL1A2 Collagen Type I Alpha 2 Chain Declarations Acknowledgments We thank Professors He Wu and Zengxiang Dong for their help offering human’s slices. Sources of Funding This work was supported by the Natural Science Foundation of China (U24A20650, 82370448, 82100492), State Key Laboratory of Frigid Zone Cardiovascular diseases, Ministry of Science and Technology, Open subject (HDHY2024010), and Special project funded by the Ministry of Science and Technology of China (2024GZkf-03). Disclosures None References Golledge J. Abdominal aortic aneurysm: update on pathogenesis and medical treatments. Nat Rev Cardiol 16 , 225-242 (2019). Cho MJ, Lee MR, Park JG. Aortic aneurysms: current pathogenesis and therapeutic targets. Exp Mol Med 55 , 2519-2530 (2023). Lu H , et al. Vascular Smooth Muscle Cells in Aortic Aneurysm: From Genetics to Mechanisms. J Am Heart Assoc 10 , e023601 (2021). Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci 123 , 4195-4200 (2010). Islam S, Watanabe H. Versican: A Dynamic Regulator of the Extracellular Matrix. J Histochem Cytochem 68 , 763-775 (2020). 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Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J Clin Invest 105 , 1605-1612 (2000). Lu H, Howatt DA, Balakrishnan A, Graham MJ, Mullick AE, Daugherty A. Hypercholesterolemia Induced by a PCSK9 Gain-of-Function Mutation Augments Angiotensin II-Induced Abdominal Aortic Aneurysms in C57BL/6 Mice-Brief Report. Arterioscler Thromb Vasc Biol 36 , 1753-1757 (2016). Kanematsu Y , et al. Pharmacologically Induced Thoracic and Abdominal Aortic Aneurysms in Mice. Hypertension 55 , 1267-1274 (2010). Oyama K , et al. Deletion of HP1γ in cardiac myocytes affects H4K20me3 levels but does not impact cardiac growth. Epigenetics Chromatin 11 , 18 (2018). Alam H , et al. HP1γ Promotes Lung Adenocarcinoma by Downregulating the Transcription-Repressive Regulators NCOR2 and ZBTB7A. Cancer Res 78 , 3834-3848 (2018). Lin Q , et al. Cystathionine γ-Lyase Attenuates Vascular Smooth Muscle Cell Senescence via Foxm1-Gas1 Pathway to Mediate Arterial Stiffness. Antioxid Redox Signal 42 , 655-671 (2025). Jiang S , et al. Hydrogen sulphide reduces hyperhomocysteinaemia-induced endothelial ER stress by sulfhydrating protein disulphide isomerase to attenuate atherosclerosis. J Cell Mol Med 25 , 3437-3448 (2021). Musto L, Smith A, Pepper C, Bujkiewicz S, Bown M. Risk factor-targeted abdominal aortic aneurysm screening: systematic review of risk prediction for abdominal aortic aneurysm. Br J Surg 111 , znae239 (2024). Chi Z , et al. Honokiol ameliorates angiotensin II-induced hypertension and endothelial dysfunction by inhibiting HDAC6-mediated cystathionine γ-lyase degradation. J Cell Mol Med 24 , 10663-10676 (2020). Bai L , et al. Angiotensin II downregulates vascular endothelial cell hydrogen sulfide production by enhancing cystathionine γ-lyase degradation through ROS-activated ubiquitination pathway. Biochem Biophys Res Commun 514 , 907-912 (2019). Chi Z , et al. Histone deacetylase 6 inhibitor tubastatin A attenuates angiotensin II-induced hypertension by preventing cystathionine γ-lyase protein degradation. Pharmacol Res 146 , 104281 (2019). Halper J. Proteoglycans and diseases of soft tissues. Adv Exp Med Biol 802 , 49-58 (2014). Wang S , et al. Postnatal deficiency of ADAMTS1 ameliorates thoracic aortic aneurysm and dissection in mice. Exp Physiol 103 , 1717-1731 (2018). Mizui Y, Yamazaki K, Kuboi Y, Sagane K, Tanaka I. Characterization of 5'-flanking region of human aggrecanase-1 (ADAMTS4) gene. Mol Biol Rep 27 , 167-173 (2000). Wang C , et al. Epigenetic Up-Regulation of ADAMTS4 in Sympathetic Ganglia is Involved in the Maintenance of Neuropathic Pain Following Nerve Injury. Neurochem Res 48 , 2350-2359 (2023). Wu W , et al. Cystathionine-γ-lyase ameliorates the histone demethylase JMJD3-mediated autoimmune response in rheumatoid arthritis. Cell Mol Immunol 16 , 694-705 (2019). van Wijnen AJ , et al. Biological functions of chromobox (CBX) proteins in stem cell self-renewal, lineage-commitment, cancer and development. Bone 143 , 115659 (2021). Zhang C , et al. Cbx3 inhibits vascular smooth muscle cell proliferation, migration, and neointima formation. Cardiovasc Res 114 , 443-455 (2018). Wang G, Xiao Q, Luo Z, Ye S, Xu Q. Functional impact of heterogeneous nuclear ribonucleoprotein A2/B1 in smooth muscle differentiation from stem cells and embryonic arteriogenesis. J Biol Chem 287 , 2896-2906 (2012). Wang S , et al. Lactate reprograms glioblastoma immunity through CBX3-regulated histone lactylation. J Clin Invest 134 , (2024). Vienneau JR, Burns CI, Boghokian A, Soti V. Endovascular Aneurysm Repair Versus Open Surgical Repair in Treating Abdominal Aortic Aneurysm. Cureus 16 , e73066 (2024). Bossone E, Eagle KA. Epidemiology and management of aortic disease: aortic aneurysms and acute aortic syndromes. Nat Rev Cardiol 18 , 331-348 (2021). Golledge J, Thanigaimani S, Powell JT, Tsao PS. Pathogenesis and management of abdominal aortic aneurysm. Eur Heart J 44 , 2682-2697 (2023). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaldata.pdf Supplementary Dataset1 SupplementalTable1.xlsx Supplementary Table 1 SupplementalTable2.xlsx Supplementary Table 2 SupplementalTable3.xlsx Supplementary Table 3 Cite Share Download PDF Status: Under Review 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. 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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-7423522","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":508398724,"identity":"f1ff733b-d46e-4e4f-96cc-1497a9a16ed3","order_by":0,"name":"Bin 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University","correspondingAuthor":false,"prefix":"","firstName":"Yaping","middleName":"","lastName":"Niu","suffix":""},{"id":508398729,"identity":"eaf5efad-ee84-404a-9b79-4c7f66c5289e","order_by":5,"name":"Ling Cheng","email":"","orcid":"","institution":"Department of Cardiology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Cheng","suffix":""},{"id":508398730,"identity":"32eb76a8-6da2-4931-ad3e-92ec897db8a8","order_by":6,"name":"Xiaodie Shao","email":"","orcid":"","institution":"State Key Laboratory of Cardiovascular Disease, Fuwai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaodie","middleName":"","lastName":"Shao","suffix":""},{"id":508398731,"identity":"31bb9f7f-b41f-42d8-a01e-d7a66c5724a8","order_by":7,"name":"Haizeng Zhang","email":"","orcid":"","institution":"Hypertension Center, Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Diseases","correspondingAuthor":false,"prefix":"","firstName":"Haizeng","middleName":"","lastName":"Zhang","suffix":""},{"id":508398732,"identity":"3a5ae287-2ae8-4f16-a36f-784edcd03a55","order_by":8,"name":"Yuan Wang","email":"","orcid":"","institution":"State Key Laboratory of Cardiovascular Disease, Fuwai Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Wang","suffix":""},{"id":508398733,"identity":"f48f7473-ff05-429b-a564-3a5b48bb98d9","order_by":9,"name":"Yuanzhen Lin","email":"","orcid":"","institution":"Department of Cardiology, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou","correspondingAuthor":false,"prefix":"","firstName":"Yuanzhen","middleName":"","lastName":"Lin","suffix":""},{"id":508398734,"identity":"46a67404-f234-40f5-8856-35a423c076ee","order_by":10,"name":"Zengxiang Dong","email":"","orcid":"https://orcid.org/0000-0001-5255-3270","institution":"The First Affiliated Hospital of Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zengxiang","middleName":"","lastName":"Dong","suffix":""},{"id":508398735,"identity":"886284e8-c22b-4f18-a0d8-72c66368b3e2","order_by":11,"name":"He Wu","email":"","orcid":"","institution":"Department of Pathology","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Wu","suffix":""},{"id":508398736,"identity":"591153c6-91cc-4102-ad02-7ce794ac03fb","order_by":12,"name":"Zhenzhen Chen","email":"","orcid":"https://orcid.org/0000-0001-9919-3997","institution":"Fuwai hospital, Chinese academy of medical science and peking union medical college","correspondingAuthor":false,"prefix":"","firstName":"Zhenzhen","middleName":"","lastName":"Chen","suffix":""},{"id":508398737,"identity":"66bee961-2c65-46db-9161-c94f2fff5b8e","order_by":13,"name":"Liming Yang","email":"","orcid":"","institution":"Department of Pathophysiology, Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liming","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-08-21 07:45:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7423522/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7423522/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90471055,"identity":"1d6a45ec-769c-450f-b585-d9fe8fcbae78","added_by":"auto","created_at":"2025-09-03 06:20:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8111658,"visible":true,"origin":"","legend":"\u003cp\u003eDownregulation of CSE in aortic tissues of AAD patients and experimental models. (A) Immunofluorescence analysis of CSE expression in human AAD specimens. Aortic tissues from 15 AAD patients were immunofluorescence stained for CSE (green) and α-smooth muscle actin (α-SMA, red; a VSMC marker). Semi-quantitative analysis revealed reduced CSE expression in the aneurysm region compared to non-AAD patient’s aorta tissues (n=12). Scale bar: 50 μm. (Mean ± SEM; n = 10–12) (B) CSE expression in VSMCs of an AngII-induced AAD model in ApoE\u003csup\u003e-/- \u003c/sup\u003emice. Immunofluorescence staining demonstrates diminished CSE levels in aneurysmal aortic segments. Scale bar: 50 μm. (Mean ± SEM; n = 8-10) (C, D) AngII suppresses CSE expression in human aortic smooth muscle cells (HASMCs). HASMCs were treated with AngII (1 μM, 24 h), and CSE mRNA (C; quantified by qRT-PCR) and protein levels (D; assessed by Western blot) were significantly downregulated. (Mean ± SEM; n = 5-6) (E, F) Consistent with human data, AngII treatment reduced CSE mRNA (E) and protein (F) expression in primary mouse aortic smooth muscle cells (mASMCs). (Mean ± SEM; n = 5-6). Intergroup differences were evaluated by unpaired two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/60e241fc4213769c06e16f50.png"},{"id":90469921,"identity":"963c4eb5-952a-4097-aa3e-609491554458","added_by":"auto","created_at":"2025-09-03 06:12:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9222218,"visible":true,"origin":"","legend":"\u003cp\u003e(A–D) Hypercholesterolemia- and AngII-induced AAD model: VSMC-specific CSE knockout mice (CSE\u003csup\u003eSMCKO\u003c/sup\u003e) and control mice (CSE\u003csup\u003eLOXP\u003c/sup\u003e) were subjected to an AAD model via liver-specific PCSK9 overexpression (inducing hypercholesterolemia), Paigen diet feeding, and AngII infusion. After 4 weeks, CSE\u003csup\u003eSMCKO\u003c/sup\u003e mice exhibited aggravated AAD formation and progression compared to controls, as demonstrated by: (A) representative aortic images and AAD incidence rate (Mean ± SEM; n = 22-25), (B) survival curve (Mean ± SEM; n = 22-25), (C) aortic diameter measured by ultrasound (Mean ± SEM; n = 12), (D) elastin degradation assessed by H\u0026amp;E and EVG staining (grading scale shown) (Mean ± SEM; n = 12-14). (E–H) BAPN plus AngII-induced AAA model for phenotypic validation: consistent with the first model, CSE\u003csup\u003eSMCKO\u003c/sup\u003e mice displayed heightened AAD severity compared to CSE\u003csup\u003eLOXP\u003c/sup\u003e mice, including: (E) increased AAA incidence (Mean ± SEM; n = 23-29), (F) elevated mortality (Mean ± SEM; n = 23-29), (G) expanded aortic diameter (Mean ± SEM; n = 8-10), and (H) enhanced elastin degradation (Mean ± SEM; n = 11). Kolmogorov-Smirnov test for data normality and unpaired two-tailed Student’s t-test for intergroup difference were used. Mantel-Cox test was used for survival curve analysis\u003c/p\u003e\n\u003cp\u003eVSMC-specific CSE deletion exacerbates AAD incidence and progression.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/d18ec23bb75326cfbdb83b60.png"},{"id":90469910,"identity":"00b6bd5c-91c1-4b56-a3e1-603bef716906","added_by":"auto","created_at":"2025-09-03 06:12:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10782760,"visible":true,"origin":"","legend":"\u003cp\u003eVSMC-specific CSE deletion promotes ADAMTS4 accumulation and versican degradation. (A) Heatmap of RNA-seq analysis showing differential expression of ADAM family genes in cultured primary CSE-knockout (KO) and wild-type (WT) VSMCs treated with AngII (1 μM, 24 h). *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (Mean ± SEM; n =5). (B) qRT-PCR validation confirmed significant upregulation of \u003cem\u003eAdamts4\u003c/em\u003e in CSE-KO VSMCs (Mean ± SEM; n = 5). (C) Western blot analysis revealed elevated ADAMTS4 protein levels and enhanced versican degradation in CSE-KO VSMCs (Mean ± SEM; n = 6). (D) Conversely, CSE overexpression in VSMCs suppressed ADAMTS4 expression and reduced versican cleavage (Mean ± SEM; n = 6). (E) In vivo, ADAMTS4 protein and the versican cleavage product (V1) were elevated in aortic VSMCs from CSE\u003csup\u003eSMCKO\u003c/sup\u003e mice compared to CSE\u003csup\u003eLOXP\u003c/sup\u003e controls in two AngII-induced AAD models (Mean ± SEM; n =5-8). (F) Increased ADAMTS4 expression was also observed in human AAD tissues (Mean ± SEM; n = 10-12). (G) In AngII-induced ApoE\u003csup\u003e−/−\u003c/sup\u003e AAD aorta, ADAMTS4 accumulation correlated with versican V1 deposition (Mean ± SEM; n = 6-9). For two-factor comparisons (CSE overexpression and AngII treatment), two-way mixed-effects ANOVA followed by Tukey’s multiple comparisons test was applied. Other two-group comparisons were analyzed by unpaired two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/819190e5a8a3e72eed1dce8a.png"},{"id":90469903,"identity":"9bac12bc-9aa4-4898-b814-42ecd90a0761","added_by":"auto","created_at":"2025-09-03 06:12:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7732697,"visible":true,"origin":"","legend":"\u003cp\u003eCSE downregulates ADAMTS4 in a CBX3-dependent manner. (A) Heatmap showing alterations in PRC1 and PRC2 gene expression in RNA-seq analysis of CSE-KO VSMCs. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 indicates significantly downregulated genes (Mean ± SEM; n =5). CBX3 protein expression was reduced in CSE-deficient VSMCs, as confirmed by Western blot (Mean ± SEM; n = 6) (B) and immunofluorescence staining (Mean ± SEM; n = 4) (C). Conversely, CSE overexpression upregulated CBX3 protein levels (Mean ± SEM; n =5) (D). Consistent with these findings, CBX3 expression was also diminished in aortic VSMCs from two distinct CSE\u003csup\u003eSMCKO\u003c/sup\u003e AAD mouse models (Mean ± SEM; n = 5-12) (E), as well as in human AAD tissues and ApoE\u003csup\u003e−/−\u003c/sup\u003e AAD models (Mean ± SEM; n =7-10) (F). Genetic knockdown of \u003cem\u003eCbx3\u003c/em\u003e via siRNA in primary CSE-KO VSMCs exacerbated ADAMTS4 upregulation and versican cleavage (Mean ± SEM; n = 5) (G), whereas Cbx3 overexpression rescued ADAMTS4 induction and versican degradation caused by CSE deficiency (Mean ± SEM; n = 5-6) (H). Data in (D) and (H) were analyzed by two-way mixed-effects ANOVA followed by Tukey’s multiple comparisons test; all other panels were assessed using an unpaired two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/af4b57e2703bab88e5761814.png"},{"id":90469911,"identity":"117d7fc0-6307-453d-966d-faff6d229f6f","added_by":"auto","created_at":"2025-09-03 06:12:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11920786,"visible":true,"origin":"","legend":"\u003cp\u003eLocal CBX3 overexpression rescues BAPN/AngII-induced AAD in CSE\u003csup\u003eSMCKO\u003c/sup\u003e mice via AAV9-mediated gene delivery. An absorbable collagen sponge-based extravascular carrier, loaded with AAV9-sm22α-Cbx3, was implanted around the abdominal aorta (between the diaphragm and renal artery) to achieve smooth muscle cell-specific CBX3 overexpression. Local CBX3 restoration attenuated AAD formation in CSE\u003csup\u003eSMCKO\u003c/sup\u003e mice, as demonstrated by: (A) Representative aortic images, (B) reduced AAD incidence (Mean ± SEM; n = 14), and (C) suppressed aortic dilation (Mean ± SEM; n = 11-14). Histological and molecular analyses further revealed that CBX3 overexpression mitigated (D) elastin degradation and collagen deposition (Mean ± SEM; n = 13), (E) versican (V1) accumulation (Mean ± SEM; n = 7-8), (F) ADAMTS4 upregulation (Mean ± SEM; n = 6), (G) VSMC apoptosis (Mean ± SEM; n = 9-10), and (H) macrophage infiltration alongside reduced MMP2/MMP9 expression (Mean ± SEM; n = 5-9). Statistical analysis: Unpaired two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/49ef89287554cf2f11c14f6e.png"},{"id":90471056,"identity":"41e37509-7c70-485f-884d-6642cd476b65","added_by":"auto","created_at":"2025-09-03 06:20:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":9446410,"visible":true,"origin":"","legend":"\u003cp\u003eCSE regulates histone modifications via CBX3 and profiling histone modifications in CSE-deficient VSMCs. (A) Western blot analysis of primary CSE-knockout VSMCs revealed reduced methylation (me3) and acetylation (ac) of H3K9 and H3K4 following stimulation with AngII or ox-LDL, whereas H3K27 methylation remained unchanged (n = 6). (B) Conversely, CSE overexpression increased H3K9me3, H3K9ac, H3K4me3, and H3K4ac levels but did not affect H3K27me3, as demonstrated by western blot (n =6). (C) Immunofluorescence staining confirmed decreased H3K9me3 and H3K9ac expression in aortic tissues from the AngII-induced AAD models with VSMC-specific CSE knockout (CSE\u003csup\u003eSMCKO\u003c/sup\u003e mice) (n = 10-13). (D) Similarly, immunofluorescence staining showed reduced H3K4me3 and H3K4ac expression in aortic tissues from two AngII-induced AAD models following CSE deletion in VSMCs (n = 5-10). (E) Western blot analysis of CSE-knockout VSMCs demonstrated that CBX3 knockdown further diminished H3K9me3/ac and H3K4me3/ac protein expression (n = 6). (F) Overexpression of CBX3 rescued the downregulation of H3K9me3/ac and H3K4me3/ac induced by CSE deletion (n =5-6). Data in (B) and (F) were analyzed by two-way mixed-effects ANOVA followed by Tukey’s multiple comparisons test. Data are presented as mean ± SEM. All other panels were analyzed using an unpaired two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/3002d7bd97a63dccaf65c39e.png"},{"id":90469928,"identity":"5436ac33-e2eb-4a6f-b3c2-2cb53d4aa6c0","added_by":"auto","created_at":"2025-09-03 06:12:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4780092,"visible":true,"origin":"","legend":"\u003cp\u003eProfiling histone modifications in CSE-deficient VSMCs. (A) Analysis of H3K9me3 in WT vs. KO VSMCs (n = 3). Upper panel: Heatmap of H3K9me3 enrichment. Middle panel: Average ChIP-seq signal profiles. Lower panel: Venn diagram depicting overlaps between H3K9me3-target genes and differentially expressed genes from RNA-seq. Right panel: GO enrichment analysis of overlapping genes. (B) Integrated analysis of H3K9ac ChIP-seq and RNA-seq, with GO enrichment of overlapping genes (n = 3). (C) Integrated analysis of H3K4me3 ChIP-seq and RNA-seq, with GO enrichment of overlapping genes (n = 3). (D\u0026amp;E) IGV tracks displaying ChIP-seq signals for H3K9me3, H3K9ac, and H3K4me3 at key genes: (D) ECM remodeling-related genes (\u003cem\u003eAdamts4\u003c/em\u003e, \u003cem\u003eMmp9\u003c/em\u003e) (n = 3), (E) \u003cem\u003eCol1a2\u003c/em\u003e (ECM-associated gene) (n = 3), Inflammatory gene \u003cem\u003eNlrp3\u003c/em\u003e (n = 3), Apoptosis-related gene \u003cem\u003eBcl2\u003c/em\u003e (n = 3). (F) qRT-PCR validation of \u003cem\u003eCol1a2\u003c/em\u003e, \u003cem\u003eNlrp3\u003c/em\u003e, and \u003cem\u003eBcl2\u003c/em\u003eexpression in CSE-KO VSMCs (n = 6). Statistical analysis: Data are presented as mean ± SEM. Data (F)were analyzed using an unpaired two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/2b86b96289a28793455e19aa.png"},{"id":90471053,"identity":"fe0e24a1-8a3f-4859-86bb-9e173eaf07f6","added_by":"auto","created_at":"2025-09-03 06:20:29","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2668946,"visible":true,"origin":"","legend":"\u003cp\u003eCBX3 forms an epigenetic complex with partner proteins. (A) Validation of CBX3-protein interactions in HEK293T cells. Plasmids encoding Flag-tagged CBX3, HA-tagged KDM2A, Myc-tagged HDAC1, His-tagged RING1, and Strep-tagged SUV39H1 were co-transfected in pairwise combinations. Protein interactions were assessed by Co-IP using tag-specific antibodies. (B) Co-IP confirmation of endogenous CBX3-protein interactions in primary VSMCs. (C) Disruption of CBX3 interactions with SUV39H1, KDM2A, and HDAC1 in CSE-deficient VSMCs (n = 3). Data represent mean ± SEM; significance was determined by unpaired two-tailed Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/86cf4f0290e0347c2fa8ed4a.png"},{"id":90469939,"identity":"d1c939b9-85c3-474e-94c2-7fdcf7fe9e2d","added_by":"auto","created_at":"2025-09-03 06:12:30","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":12860416,"visible":true,"origin":"","legend":"\u003cp\u003eLocal overexpression of CSE or CBX3 via an extravascular carrier attenuates AAD incidence and progression. (A-E) A collagen-based absorbable extravascular carrier was implanted on the abdominal aorta and loaded with AAV9-sm22α-CSE or AAV9-sm22α-CBX3 to achieve localized overexpression of CSE or CBX3 in the aortic wall. This intervention mitigated AngII-induced AAD formation and progression in ApoE\u003csup\u003e-/- \u003c/sup\u003emice, as evidenced by: (A) Representative aortic images and incidence rate of AAD ( n = 12). (B) Reduced abdominal aortic diameter (n = 12). (C) Attenuated elastin degradation (n = 12). (D) Decreased ADAMTS4 accumulation (n = 12). (E) Suppressed versican cleavage (n = 10). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/a1ac243ddd8898e98df5614a.png"},{"id":90472804,"identity":"c64f1e95-7609-467a-8bdc-fc56c0c8eeff","added_by":"auto","created_at":"2025-09-03 06:37:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":71607149,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/9c42a9b9-8472-4534-b43d-4fb8a236ced5.pdf"},{"id":90469900,"identity":"24f88f5c-2131-4fc5-8605-2666cdf7d92c","added_by":"auto","created_at":"2025-09-03 06:12:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3616634,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Dataset1\u003c/p\u003e","description":"","filename":"Supplementaldata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/c9ca21851da03b5c6ba87484.pdf"},{"id":90471862,"identity":"60c374cb-9f50-4f41-8073-671300152883","added_by":"auto","created_at":"2025-09-03 06:28:29","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10424,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 1\u003c/p\u003e","description":"","filename":"SupplementalTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/c5e16644de76ebff4e222756.xlsx"},{"id":90469904,"identity":"8fcb504d-ff6a-457d-a181-81f2074bbb1b","added_by":"auto","created_at":"2025-09-03 06:12:28","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11188,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 2\u003c/p\u003e","description":"","filename":"SupplementalTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/c1839b5817a77a8b12bc8081.xlsx"},{"id":90469919,"identity":"d47e9aa3-e275-423d-8567-c51d7fedb7ce","added_by":"auto","created_at":"2025-09-03 06:12:29","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":21836,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Table 3\u003c/p\u003e","description":"","filename":"SupplementalTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7423522/v1/d3510cef2e72c8b89904d3d9.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Chromobox 3 Assembles a Novel Epigenetic Complex Contributing to Cystathionine γ-lyase–mediated Protection Against Aortic Aneurysm/dissection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAortic aneurysm/dissection (AAD) is a chronic aortic disorder characterized by progressive weakening and dilation of the vessel wall, frequently culminating in fatal rupture and accounting for an estimated 150,000\u0026ndash;200,000 deaths annually worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Hallmark pathological features include vascular smooth muscle cell (VSMC) loss, extracellular matrix (ECM) degradation (particularly elastin and collagen), proteoglycan accumulation, and localized inflammation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. VSMCs are central to maintaining aortic integrity through ECM synthesis, regulation of cell death, and modulation of inflammatory responses\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe aortic ECM is a dynamic network composed of fibrous proteins (collagen, elastin, fibrillins) and proteoglycans such as versican and aggrecan \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Pathological ECM remodeling in AAD is driven by proteases, notably matrix metalloproteinases (MMPs), which degrade structural proteins, and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteoglycanases\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Versican, a large chondroitin sulfate proteoglycan predominantly expressed in VSMCs\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, plays a critical role in vascular homeostasis \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Versican accumulation is consistently observed in human and experimental AAD \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Within the ADAMTS family, six members (ADAMTS1, 4, 5, 9, 15, 20) possess proteoglycanase activity\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. ADAMTS5 exhibits the highest activity against versican, followed by ADAMTS4 and ADAMTS1\u003csup\u003e12, 13\u003c/sup\u003e. Notably, ADAMTS4\u0026mdash;upregulated in both clinical and experimental AAD\u0026mdash;cleaves versican at the Glu441-Ala442 site\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, whereas ADAMTS5 is paradoxically downregulated \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Genetic studies confirm this functional divergence: ADAMTS4 deficiency is protective against aneurysm, while loss of ADAMTS1 or ADAMTS5 accelerates disease\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Together, these findings underscore the importance of versicanolysis in ECM remodeling and AAD pathogenesis.\u003c/p\u003e\u003cp\u003eHydrogen sulfide (H₂S), an endogenous gasotransmitter synthesized mainly by cystathionine γ-lyase (CSE), exerts vasoprotective effects via protein sulfhydration\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Clinical and experimental studies consistently report reduced CSE expression and H₂S bioavailability in AAD\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Endothelial-specific CSE deficiency exacerbates dissection through enhanced endoplasmic reticulum stress, a process reversed by H₂S-mediated sulfhydration of protein disulfide isomerase\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. H₂S also inactivates MMP-2 and MMP-9 by sulfhydrating their cysteine-switch motifs\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. While VSMCs produce MMPs, major sources of MMP-2 and MMP-9 in dissection are fibroblasts/leukocytes and macrophages, respectively\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Importantly, VSMCs are the predominant producers of vascular H₂S\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Thus, although H₂S-mediated MMP inactivation may partly account for CSE/H₂S vasoprotection, the broader regulatory mechanisms, particularly concerning ADAMTS proteases and proteoglycan turnover, remain poorly defined.\u003c/p\u003e\u003cp\u003eHere, we show that VSMC-specific CSE deletion aggravates AAD through epigenetic dysregulation of the ADAMTS4\u0026ndash;versican axis. Mechanistically, CSE deficiency suppresses chromobox 3 (CBX3), disrupting histone H3K4/H3K9 methylation and acetylation, thereby derepressing \u003cem\u003eAdamts4\u003c/em\u003e transcription. We further identify CBX3 as a scaffold for a previously unrecognized histone-modifying complex integrating methylation and acetylation pathways. Finally, local delivery of CSE or CBX3 via absorbable extravascular carrier mitigates AAD progression, highlighting a promising translational strategy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe data, research methods, and study materials utilized in this study are available from the corresponding authors on reasonable request. Detailed Expanded Methods section are provided in the Supplemental Material.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHuman Aortic Samples\u003c/h2\u003e\u003cp\u003eThe human aortic tissues were collected in compliance with the Decleration of Helsinki. The AAD aortic samples were collected from patients who suffered aneurysmectomy and vascular transplantation. The non-AAD samples were from the Biospeciman Bank of the First Affiliated Hospital of Harbin Medical University. The baseline characteristics of the patients are presented in Supplementary Table\u0026nbsp;1. The study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University (2025141).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAnimal Study and AAD Models\u003c/h3\u003e\n\u003cp\u003e Animal experiments were approved by Institute of Animal Care and Use Committee of Fuwai Hospital, Chinese Academy of Medical Sciences (FW-2020-0059), and and performed in accordance with the animal experiment guidline. CSE\u003csup\u003eSMCKO\u003c/sup\u003e knockout mice generated by CSE\u003csup\u003eLOXP\u003c/sup\u003e (containing loxP sites flanking exons 2 and 3) hybridzation with Tagln-Cre mice as our previous study\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. ApoE knockout mice (ApoE\u003csup\u003e-/-\u003c/sup\u003e) and C57BL/6J mice were purchased by Charles River Laboratories (Beijing, China).\u003c/p\u003e\u003cp\u003eAngII-induced AAD model was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Briefly, 8- to 10-week-old male ApoE\u003csup\u003e-/-\u003c/sup\u003e mice were subcutaneously implanted with minipumps (Alzet, Model 2004) to infuse AngII (1000ng/kg/min) for 4 weeks while being maintained on a Paigen diet to induce AAD.\u003c/p\u003e\u003cp\u003eThe PCSK9/AngII-induced AAD model was established following established protocols\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Briefly, 8- to 10-week-old mice (CSE\u003csup\u003eLOXP\u003c/sup\u003e, CSE\u003csup\u003eSMCKO\u003c/sup\u003e) received intravenous injection of recombinant adeno-associated virus (rAAV8-HCRApoE/hAAT-D377Y-mPCSK9; 5\u0026times;10\u0026sup1;\u0026sup1; vector genome copies in 150 \u0026micro;l sterile saline) while being maintained on a Paigen diet to induce hyperlipidemia. One week post-AAV administration, AngII (1,000 ng/kg/min) was continuously delivered via subcutaneous osmotic minipump for 4 weeks to promote AAD development.\u003c/p\u003e\u003cp\u003eThe β-aminopropionitrile (BAPN)/AngII-induced AAD model was established as previously described\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Briefly, 8- to 10-week-old mice received concurrent administration of AngII (1,000 ng/kg/min) via osmotic minipump and BAPN (150 mg/kg/day) in drinking water for 4 weeks to induce aortic aneurysm formation.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean with standard error of the mean (SEM). Differences between two groups were evaluated with unpaired 2-tailed T-test (normal distribution), Mann-Whitney test (non-normal distribution), or Fisher's exact test (categorical data). For more groups, data were compared by one-way ANOVA followed by post-hoc analysis. Comparisons including two factors were performed by two-way ANOVA, with repeated measures on the same animals or cells analyzed via two-way mixed-effects ANOVA. All statistical analysis involved using GraphPad Prism v10.1.2. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eDown-regulated VSMC CSE expression in AAD\u003c/h2\u003e\u003cp\u003eTo examine the alterations of CSE expression in VSMCs within AAD, we performed immunofluorescence staining. Compared with healthy aortic tissues, CSE staining (green) in α-SMA\u0026ndash;positive VSMCs (red) was markedly reduced in AAD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This observation is consistent with prior reports demonstrating decreased CSE expression in diseased aortic tissues, further supported by Western blot analyses\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In the aorta of ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, CSE expression was detectable in both endothelial cells and VSMCs, but significantly decreased in VSMCs within the AngII-induced AAD model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;1). In vitro, AngII treatment notably suppressed both CSE mRNA and protein expression in human and mouse aortic smooth muscle cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;F). Collectively, evidence from human samples, animal models, and cultured cells demonstrates that VSMC-derived CSE expression is downregulated during AAD development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eVSMC-Specific Deletion of CSE Promotes AAD Progression\u003c/h2\u003e\u003cp\u003eTo determine the functional role of CSE in AAD pathogenesis, we assessed phenotypic changes in two AngII-induced AAD mouse models. In the first model, AAD was established by overexpressing PCSK9 in the liver using AAV8-D377Y-mPCSK9 combined with a Paigen diet to induce hypercholesterolemia, followed by AngII infusion for four weeks. VSMC-specific CSE deletion (validated in Supplementary Fig.\u0026nbsp;2A, B) significantly increased AAD incidence (95.4% vs. 48%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), mortality (22% vs. 7%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), abdominal aortic diameter (1.96 mm vs. 1.42 mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and elastin degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) compared with controls.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further confirm these results, we employed a second AAD model combining BAPN administration with AngII infusion. Consistent with findings from the AAV-PCSK9\u0026thinsp;+\u0026thinsp;AngII model, CSE deletion in VSMCs markedly elevated AAD incidence (95.6% vs. 34.4%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), mortality (60% vs. 16%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), aortic diameter (1.28 mm vs. 0.92 mm; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), and elastin degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Moreover, CSE-deficient VSMCs displayed enhanced apoptosis (Supplementary Fig.\u0026nbsp;2C) and increased expression of MMP2 and MMP9 (Supplementary Fig.\u0026nbsp;2D). These results strongly indicate that CSE deficiency in VSMCs accelerates AAD initiation and progression.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCSE Deficiency in VSMCs Induces ADAMTS4 Accumulation and Versican Degradation\u003c/h3\u003e\n\u003cp\u003eECM remodeling is a hallmark of AAD pathogenesis, driven by MMP-mediated degradation of collagen and elastin and ADAMTS-mediated degradation of proteoglycans. To elucidate the role of CSE in ECM regulation, we performed RNA sequencing of AngII-stimulated VSMCs. Transcriptomic analysis (GEO: GSE290627) revealed significant alterations in MMP and ADAMTS family members: \u003cem\u003eMmp9\u003c/em\u003e, \u003cem\u003eMmp10\u003c/em\u003e, \u003cem\u003eMmp13\u003c/em\u003e, \u003cem\u003eAdamts3\u003c/em\u003e, and \u003cem\u003eAdamts4\u003c/em\u003e were upregulated, whereas \u003cem\u003eAdamts9\u003c/em\u003e and \u003cem\u003eAdamts10\u003c/em\u003e were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;3A). qRT-PCR validated these results, except for \u003cem\u003eAdamts10\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Fig.\u0026nbsp;3B), in CSE-deficient VSMCs following AngII exposure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eProtein analysis confirmed that CSE knockout augmented ADAMTS4 expression and enhanced versican degradation in VSMCs upon AngII (1 \u0026micro;M) stimulation for 24 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Conversely, CSE overexpression suppressed ADAMTS4 expression and reduced versican degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Pathological staining further demonstrated elevated ADAMTS4 levels in α-SMA\u0026ndash;positive aortic VSMCs and increased accumulation of the versican cleavage fragment V1 in CSE\u003csup\u003eSMCKO\u003c/sup\u003e compared with CSE\u003csup\u003eLOXP\u003c/sup\u003e mice across both AAD models (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eImportantly, increased ADAMTS4 expression was also detected in VSMCs from human AAD tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) and in the AngII-induced ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), coinciding with pronounced versican degradation. These findings, consistent with previous studies\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, underscore a critical role of the ADAMTS4\u0026ndash;versican axis in mediating CSE-regulated ECM remodeling during AAD pathogenesis.\u003c/p\u003e\n\u003ch3\u003eCBX3 Negatively Regulates ADAMTS4 Expression\u003c/h3\u003e\n\u003cp\u003ePolycomb repressive complexes (PRCs), comprising PRC1 and PRC2, act as key epigenetic regulators by modifying histones to suppress gene transcription. To identify PRC components involved in \u003cem\u003eAdamts4\u003c/em\u003e regulation, we analyzed downregulated genes from RNA-seq data and validated them by qRT-PCR. Both \u003cem\u003eCbx3\u003c/em\u003e and Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit (\u003cem\u003eEzh2\u003c/em\u003e) were reduced following CSE deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;4A). Functional studies revealed that siRNA-mediated knockdown of \u003cem\u003eEzh2\u003c/em\u003e decreased \u003cem\u003eAdamts4\u003c/em\u003e transcription, whereas Cbx3 knockdown markedly increased \u003cem\u003eAdamts4\u003c/em\u003e expression (Supplementary Fig.\u0026nbsp;4B, C), indicating a repressive role of CBX3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWestern blot analysis confirmed reduced CBX3 protein levels after CSE deletion under AngII or ox-LDL stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Immunofluorescence demonstrated that CBX3 predominantly localized in the nucleus but was diminished in CSE-deficient VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), whereas CSE overexpression enhanced CBX3 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In vivo, CBX3 expression was significantly reduced in VSMCs of CSE\u003csup\u003eSMCKO\u003c/sup\u003e mice compared with controls in two AAD models (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Similar downregulation was observed in VSMCs from AAA patients and ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eTo define its functional role, primary VSMCs from CSE-knockout mice were studied. CBX3 knockdown further elevated ADAMTS4 accumulation and versican cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), while CBX3 overexpression suppressed both (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). These findings establish CBX3 as a nuclear mediator linking endogenous CSE/H₂S signaling to ADAMTS4 regulation.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eCBX3 Overexpression Attenuates CSE Deficiency\u0026ndash;Induced AAD\u003c/h2\u003e\u003cp\u003eTo determine whether restoring CBX3 could mitigate AAD progression driven by CSE deficiency, we developed a localized gene delivery system. A collagen-based absorbable \u0026ldquo;extravascular stent-like carrier\u0026rdquo; was implanted around the superior renal artery, adsorbing AAV9-sm22α-Cbx3 to achieve targeted CBX3 overexpression; AAV9-sm22α-GFP served as control (Supplementary Fig.\u0026nbsp;5). One week later, mice were subjected to BAPN\u0026thinsp;+\u0026thinsp;AngII to induce AAD.\u003c/p\u003e\u003cp\u003eAmong survivors (14/group), CBX3 overexpression significantly reduced AAD incidence (4/14 vs. 12/14; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B), aortic dilation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), elastin degradation, and collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Moreover, pathological hallmarks of AAD progression\u0026mdash;including versican cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), ADAMTS4 accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), VSMC apoptosis (TUNEL staining, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), macrophage infiltration (CD68 positive cells), and elevated MMP2/9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH)\u0026mdash;were all attenuated by CBX3 overexpression. Collectively, these data demonstrate that CBX3 restoration rescues the exacerbated AAD phenotype in CSE-deficient mice, primarily via the CBX3\u0026ndash;ADAMTS4\u0026ndash;versican signaling axis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eHistone Modifications Mediate CBX3\u0026rsquo;s Response to CSE Deletion\u003c/h2\u003e\u003cp\u003eCBX3 is tightly linked to histone modifications across various cell types\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. To test whether these modifications contribute to CBX3-mediated regulation, we assessed histone marks under CSE deletion. AngII or ox-LDL stimulation in CSE-deficient VSMCs reduced H3K9me3, H3K9ac, H3K4me3, and H3K4ac, while H3K27me3 remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Conversely, CSE overexpression enhanced H3K4/9 methylation and acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In vivo, CSE\u003csup\u003eSMCKO\u003c/sup\u003e mice exhibited reduced H3K9me3/ac and H3K4me3/ac levels in the aorta across two AngII-induced AAA models (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). Similar reductions were confirmed in human AAA tissues (Supplementary Fig.\u0026nbsp;6A) and ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (Supplementary Fig.\u0026nbsp;6B), suggesting that impaired H3K4/9 methylation/acetylation represents a common epigenetic mechanism in AAD. CBX3 was further implicated in this process: knockdown in CSE-deficient VSMCs aggravated loss of H3K4/9 methylation/acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), whereas CBX3 overexpression restored these modifications under AngII stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These results indicate that CBX3 is a key mediator of CSE-induced histone modifications.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eCSE\u0026ndash;CBX3\u0026ndash;Histone Modification Axis in AAD\u003c/h2\u003e\u003cp\u003eTo delineate the molecular mechanisms, we performed ChIP-seq and RNA-seq integration. CSE deletion increased H3K9me3 enrichment near transcription start sites (TSS\u0026thinsp;\u0026plusmn;\u0026thinsp;3 kb; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA), affecting 355 genes (92 upregulated, 263 downregulated). GO and KEGG analyses revealed enrichment in ECM regulation, apoptosis, VSMC function, and inflammation pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;7). Conversely, CSE deletion reduced H3K9ac and H3K4me3 enrichment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, C). IGV profiles highlighted decreased histone modifications in ECM-related (\u003cem\u003eAdamts4\u003c/em\u003e, \u003cem\u003eMmp9\u003c/em\u003e, \u003cem\u003eCol1a2\u003c/em\u003e), inflammation-related (\u003cem\u003eNlrp3\u003c/em\u003e), and apoptosis-related (\u003cem\u003eBcl2\u003c/em\u003e) genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, E). qRT-PCR validated upregulation of \u003cem\u003eAdamts4\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), \u003cem\u003eMmp9\u003c/em\u003e (Supplementary Fig.\u0026nbsp;3B), and \u003cem\u003eNlrp3\u003c/em\u003e, alongside downregulation of \u003cem\u003eCol1a2\u003c/em\u003e and \u003cem\u003eBcl2\u003c/em\u003e in CSE-deficient VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These findings collectively demonstrate that the CSE\u0026ndash;CBX3 axis orchestrates histone modifications at H3K4/9, thereby regulating ECM remodeling, apoptosis, and inflammation pathways central to AAD development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eCBX3 as a Component of a Novel Epigenetic Regulatory Complex\u003c/h2\u003e\u003cp\u003eCBX3, a heterochromatin protein 1 (HP1)\u0026ndash;interacting factor, is known to function as an H3K9me3 methyl-reader; however, its role in modulating other histone marks such as H3K9ac, H3K4me3, and H3K4ac remains unclear. To identify CBX3-binding partners, we performed immunoprecipitation (IP) followed by proteomic analysis. SDS-PAGE with silver staining (Supplementary Fig.\u0026nbsp;8A) revealed distinct protein bands, which were analyzed by mass spectrometry, identifying 405 potential CBX3 interactors (Supplementary Table\u0026nbsp;3). GO and KEGG analyses showed significant enrichment in histone binding and modification pathways (Supplementary Fig.\u0026nbsp;8B).\u003c/p\u003e\u003cp\u003eFor validation, plasmids encoding Flag-tagged CBX3, HA-tagged KDM2A, Myc-tagged HDAC1, His-tagged RING1, and Strep-tagged SUV39H1 were co-transfected in random pairs into HEK293T cells. Co-immunoprecipitation confirmed CBX3 interactions with RING1, SUV39H1, KDM2A, and HDAC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). These interactions were further validated in primary mouse aortic VSMCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). Notably, CSE deletion disrupted CBX3 interactions with KDM2A and SUV39H1, while strengthening its association with HDAC1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Collectively, these findings indicate that CBX3 forms a dynamic epigenetic regulatory complex with SUV39H1, KDM2A, HDAC1, and RING1, responsive to changes in the CSE/H₂S system, thereby contributing to AAD pathogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eLocal Overexpression of CSE or CBX3 Attenuates AAD Development\u003c/h2\u003e\u003cp\u003eTo assess the therapeutic potential of CSE or CBX3 restoration, we implanted a collagen sponge\u0026ndash;based absorbable extravascular stent around the abdominal aorta (between the diaphragm and renal artery) loaded with AAV9-sm22α-CSE or AAV9-sm22α-CBX3. ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice underwent AngII infusion one week after implantation. Four weeks later, both CSE and CBX3 overexpression markedly reduced AAD incidence (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA), aortic diameter (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB), elastin degradation, and collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). Additionally, apoptotic cell counts (TUNEL, Supplementary Fig.\u0026nbsp;9A), macrophage infiltration (CD68 staining), and MMP2/MMP9 expression (Supplementary Fig.\u0026nbsp;9B) were significantly decreased in both treatment groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eConsistent with these phenotypic changes, CSE or CBX3 overexpression also reduced ADAMTS4 accumulation and versican cleavage in the aortic media (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD, E). Interestingly, CSE overexpression partially restored CBX3 expression to levels comparable with direct CBX3 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD), whereas CBX3 overexpression did not affect CSE expression (Supplementary Fig.\u0026nbsp;9A), confirming CBX3 as a downstream effector of CSE in vivo.\u003c/p\u003e\u003cp\u003eTogether, these findings demonstrate that targeted, VSMC-specific overexpression of CSE or CBX3 via AAV-based delivery using an absorbable extravascular carrier effectively suppresses AAD progression, highlighting a novel therapeutic strategy based on the CSE\u0026ndash;CBX3\u0026ndash;ADAMTS4 axis.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eVSMCs are the main producers of CSE and H₂S in arterial tissues, regulating VSMC functions such as contraction, proliferation, migration, apoptosis and senescence\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. H₂S influences key proteins via sulfhydration, inhibiting MMP2/MMP9 activity\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and reducing endoplasmic reticulum stress\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, thereby modulating AAD progression. However, direct evidence linking VSMC-specific CSE/H₂S signaling to AAD remains limited. In this study, we observed reduced CSE expression in VSMCs from AAD patients and murine models. Using a conditional knockout mouse model, we found that CSE deletion in VSMCs worsened AAD in two AngII-induced models. Mechanistically, CSE loss disrupted the CBX3 epigenetic complex, altering histone modifications and promoting \u003cem\u003eAdamts4\u003c/em\u003e transcription, and dysregulation of \u003cem\u003eMmp9\u003c/em\u003e, \u003cem\u003eCol1a2\u003c/em\u003e, \u003cem\u003eNlrp3\u003c/em\u003e, and \u003cem\u003eBcl2\u003c/em\u003e. These changes exacerbated versican cleavage, collagen/elastin degradation, inflammation, and apoptosis, accelerating AAD development (Supplementary Fig.\u0026nbsp;9).\u003c/p\u003e\u003cp\u003ePrevious studies have shown reduced aortic CSE expression in AAA and aortic dissection patients, as well as in the aortic endothelium of AngII-induced ApoE\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice\u003csup\u003e20, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Our study further confirms CSE downregulation in VSMCs of AAD patients and an AngII-induced mouse model using immunofluorescence staining. Risk factors for AAD, such as aging (including cell senescence and senility), male gender, hypertension, hypercholesterolemia, and diabetes (high glucose)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, Notably, several studies have reported that high glucose levels, hypercholesterolemia, are linked to decreased VSMC CSE expression\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. AngII, a key AAD inducer, directly suppresses CSE in VSMCs and endothelial cells \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, via transcriptional repression by the ZEB2-HDAC1-NuRD complex and post-translational including HDAC6-mediated acetylation at K73 and ubiquitination at K48, leading to CSE degradation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. These findings highlight how risk factors accelerate CSE reduction in aortic VSMCs, contributing to AAD pathogenesis and progression.\u003c/p\u003e\u003cp\u003eThe histopathological hallmark of ECM remodeling in AAD is characterized by the degradation and disorganization of elastic and collagen fibers, along with proteoglycan accumulation \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Among the major large proteoglycans, versican and aggrecan play crucial roles in maintaining the reversible compressive structure of the aortic wall, regulating VSMC homeostasis, and are notably upregulated in thoracic aortic aneurysm and dissection (TAAD) \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The ADAMTS family members (ADAMTS1, ADAMTS4, and ADAMTS5) exhibit proteolytic activity toward aggrecan and versican\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, with their protein expression significantly elevated in TAAD patients\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Genetic studies in mice reveal divergent roles for these proteases: \u003cem\u003eAdamts1\u003c/em\u003e heterozygosity exacerbates high-fat diet plus AngII-induced aortic events but attenuates BAPN plus AngII-induced pathology\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Deletion of the \u003cem\u003eAdamts5\u003c/em\u003e catalytic domain enhances AngII-induced ascending aortic dilation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Global \u003cem\u003eAdamts4\u003c/em\u003e knockout mitigates AngII-induced AAD formation, versican degradation, elastic fiber destruction, macrophage infiltration, and VSMC apoptosis\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. In our current study, smooth muscle cell-specific CSE knockout mice exhibited exacerbated AngII-induced AAD incidence and progression, correlating with ADAMTS4 accumulation and heightened versican degradation. Consistent with prior findings on ADAMTS4-versican dysregulation, we observed concomitant increases in elastic fiber degradation, macrophage infiltration, and VSMC apoptosis, mirroring the phenotypic consequences of \u003cem\u003eAdamts4\u003c/em\u003e deletion\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. RNA-seq analysis further confirmed that \u003cem\u003eAdamts1\u003c/em\u003e and \u003cem\u003eAdamts5\u003c/em\u003e expression remained unchanged in CSE-deficient VSMCs compared to wild-type controls. These findings collectively implicate ADAMTS4-versican axis dysregulation as a key mechanism underlying ECM remodeling, inflammatory responses, and cell death in AAD pathogenesis, mediated by VSMC CSE/H₂S signaling. Importantly, human AAD samples corroborated these results, demonstrating elevated ADAMTS4 expression and versican degradation\u0026mdash;phenotypes also observed in TAAD\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and traditional murine AAD models. Therefore, ADAMTS4 upregulation represents a conserved pathophysiological mechanism in AAD, suggesting that targeted inhibition of this protease may hold therapeutic potential for preventing or treating AAD.\u003c/p\u003e\u003cp\u003eADAMTS4 transcription is bidirectionally regulated by SP1/AP-2α (activators) and nuclear factor I (NFI)/histone H4 deacetylation (repressors) \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. While CSE/H\u003csub\u003e2\u003c/sub\u003eS inhibits SP1 via sulfhydration-mediated suppression of Jumonji domain-containing protein 3 and MMP2\u003csup\u003e22, 40\u003c/sup\u003e, its effects on AP-2α/NFI/H4 remain unknown. Here, we identified CBX3 as a novel negative regulator of \u003cem\u003eAdamts4\u003c/em\u003e transcription. CBX3 levels were significantly reduced in both CSE-deficient VSMCs/aortic tissues and human/murine AAA specimens. Functional studies demonstrated that localized CBX3 overexpression in CSE-deficient VSMCs attenuated AAD progression, concomitant with decreased ADAMTS4 expression and versican cleavage. These findings establish CBX3 as a previously unidentified transcriptional repressor in the ADAMTS4 regulatory network, providing new mechanistic insights into AAD pathogenesis.\u003c/p\u003e\u003cp\u003eThe CBX family proteins are canonical components of PRC1, which is a well-characterized transcriptional repressor primarily mediating gene silencing through H2AK119 ubiquitination\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. CBX3 exhibits dual functions as both a PRC1 component and an H3K9me3 reader through its interaction with HP1 complexes and SUV39H1/2 methyltransferases\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. It regulates diverse processes including VSMC proliferation and migration (via Notch3 signaling)\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, interfering artery development (via smooth muscle cells differentiation) \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, and H4K20me3-dependent cardiac growth\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Recent studies show CBX3 recruits EP300 to promote histone lactylation\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In this study, we identify CBX3 as a specific regulator of H3K9me3/ac and H3K4me3/ac, but not H3K27me3. Proteomic and Co-IP analyses reveal CBX3 forms a novel epigenetic complex with SUV39H1, KDM2A, HDAC1, and RING1. These interactions suggest the formation of a novel histone-modifying complex centered around CBX3. ChIP-seq also demonstrates CSE-induced CBX3 downregulation alters these marks at loci involved in ECM remodeling, apoptosis, and inflammation - key pathways in AAD pathogenesis. We propose CBX3 serves as a scaffold coordinating methylase/demethylase (SUV39H1/KDM2A), acetyltransferase/deacetylase (EP300/HDAC1), and ubiquitin ligase (RING1) activities, forming a transcriptional repressor module that drives AAD development through integrated histone modification control.\u003c/p\u003e\u003cp\u003eOpen surgery (OS) and endovascular aneurysm repair (EVAR) are the primary therapeutic strategies for AAD. Short-term outcomes favor EVAR due to its advantages, including shorter operation times, reduced intraoperative blood loss, decreased need for blood transfusions, and lower rates of postoperative mechanical ventilation\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. However, long-term mortality rates do not significantly differ between OS and EVAR, likely because EVAR requires more frequent secondary interventions\u0026mdash;often due to graft-related complications such as endoleaks\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. To address these limitations, we developed a novel \"extravascular stent-like carrier\" composed of a collagen sponge, which offers several key benefits: (1) prevention of aneurysm rupture, (2) biodegradability, and (3) the capacity to deliver therapeutic agents (e.g., viruses or drugs) for localized treatment. In our approach, the carrier was implanted around the abdominal aorta between the diaphragm and renal artery\u0026mdash;the most susceptible site for aneurysm formation in an AngII-induced AAD mouse model. This placement effectively reduced aneurysm rupture. Additionally, we loaded the carrier with AAV9 vectors to overexpress CSE or CBX3 in VSMCs, which significantly attenuated AngII-induced AAD incidence and progression. Collectively, our findings suggest that implantation of an extravascular stent combined with localized gene therapy may represent a promising alternative therapeutic strategy for AAD.\u003c/p\u003e\u003cp\u003eLimitations of the current study include the following: 1) Lack of VSMC-specific CBX3 inducible knockout mice. These mice would have been valuable to confirm whether the protective effects of H\u003csub\u003e2\u003c/sub\u003eS donors on AAD are mediated through CBX3. 2) The study did not explore how CSE/H\u003csub\u003e2\u003c/sub\u003eS signaling regulates CBX3 expression or its regulatory model of the novel epigenetic complex. 3) Only the AngII-induced AAD model was used; other established models, such as elastase-induced or CaCl2-induced aortic aneurysm, were not examined. 4) Key translational questions of extravascular stent challenges in large animals remain unresolved, including material optimization, biomechanical parameters, and therapeutic efficacy in preclinical large-animal AAD models.\u003c/p\u003e\u003cp\u003eIn summary, this study elucidates the critical role of VSMC-derived CSE/H\u003csub\u003e2\u003c/sub\u003eS in the pathogenesis of AAD and uncovers a novel epigenetic regulatory mechanism governing ECM remodeling, cellular apoptosis, and inflammatory responses in AAD progression. Furthermore, we identified a previously unrecognized epigenetic complex composed of CBX3, SUV39H1, KDM2A, HDAC1, RING1, and HAT\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, which collectively modulate the methylation and acetylation of histone H3K9 and H3K4 to regulate gene expression. From a translational perspective, we developed an innovative therapeutic approach combining an extravascular stent with localized transgene delivery, offering a potential strategy for AAD intervention. This dual-component system not only provides structural support to attenuate aneurysm progression but also enables targeted modulation of key molecular pathways implicated in AAD pathogenesis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"577\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eAAA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eAbdominal Aortic Aneurysm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eCSE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eCystathionine \u0026gamma; lyase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eHydrogen Sulfide\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eVSMCs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eVascular Smooth Muscle Cells\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eECM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eExtracellular Matrix\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eADAMTS\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eA Disintegrin-like and Metalloproteinase with Thrombospondin Motifs\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eCBX3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eChromobox 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eAAV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eAdeno-Associated Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003ePCSK9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eProprotein Convertase Subtilisin/Kexin type 9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eApoE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eApolipoprotein E\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003ePRC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003ePolycomb Repressive Complex\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eBAPN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003e\u0026beta;-aminopropionitrile\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eChIP-seq\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eChromatin Immunoprecipitation sequencing\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eTSS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eTranscription start site\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eGO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eGene Ontology\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eKEGG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eHP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eHeterochromatin Protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eMMP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eMatrix Metalloproteinases\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eNLRP3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eNOD-like receptor protein family pyrin domain containing 3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eAAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eAortic aneurysm and dissection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eTAAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eThoracic aortic aneurysm and dissection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eHAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eHistone acetyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eOS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eOpen surgery\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eEVAR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eEndovascular aneurysm repair\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eH3K4/9me3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eHistone 3 lysine 4/9 trimethylation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eH3K4/9ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eHistone 3 lysine 4/9 acetylation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eH3K27me3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eHistone 3 lysine 27 trimethylation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003e\u0026alpha;-SMA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003e\u0026alpha;-smooth muscle actin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eEZH2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eEnhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eox-LDL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eoxidized Low-Density Lipoprotein\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eHDAC1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eHistone Deacetylase 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eKDM2A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eLysine Demethylase 2A\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eSUV39H1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eSUV39H1 Histone Lysine Methyltransferase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eRING1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eRing Finger Protein 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eBCL2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eBCL2 Apoptosis Regulator\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.0243%;\"\u003e\n \u003cp\u003eCOL1A2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81.9757%;\"\u003e\n \u003cp\u003eCollagen Type I Alpha 2 Chain\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Professors He Wu and Zengxiang Dong for their help offering human\u0026rsquo;s slices.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eSources of Funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of China (U24A20650, 82370448, 82100492), State Key Laboratory of Frigid Zone Cardiovascular diseases, Ministry of Science and Technology, Open subject (HDHY2024010), and Special project funded by the Ministry of Science and Technology of China (2024GZkf-03).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eDisclosures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eGolledge J. 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Epidemiology and management of aortic disease: aortic aneurysms and acute aortic syndromes. \u003cem\u003eNat Rev Cardiol\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 331-348 (2021).\u003c/li\u003e\n \u003cli\u003eGolledge J, Thanigaimani S, Powell JT, Tsao PS. Pathogenesis and management of abdominal aortic aneurysm. \u003cem\u003eEur Heart J\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 2682-2697 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Abdominal aortic aneurysm, Cystathionine γ lyase, Hydrogen sulfide, Vascular smooth muscle cells, A disintegrin-like and metalloproteinase with thrombospondin motifs 4, Chromobox 3","lastPublishedDoi":"10.21203/rs.3.rs-7423522/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7423522/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHydrogen sulfide (H₂S), generated by cystathionine γ-lyase (CSE), exerts protective effects against aortic aneurysm/dissection (AAD). Proteoglycans, major extracellular matrix (ECM) components synthesized by vascular smooth muscle cells (VSMCs), preserve aortic wall integrity but also contribute to AAD pathogenesis. The mechanisms linking VSMC-derived CSE/H₂S to proteoglycan regulation in AAA remain undefined. Here, we identified reduced CSE expression in VSMCs (α-SMA⁺) from human AAD tissues and murine models. VSMC-specific CSE deletion (CSE\u003csup\u003eSMCKO\u003c/sup\u003e) exacerbated AngII-induced AAD, with increased ADAMTS4 expression and versican degradation. Mechanistically, CSE loss suppressed CBX3, releasing \u003cem\u003eAdamts4\u003c/em\u003e transcriptional repression. Conversely, CBX3 overexpression ameliorated AAD in CSE\u003csup\u003eSMCKO\u003c/sup\u003e mice. CBX3 formed an epigenetic complex with SUV39H1, KDM2A, HDAC1, and RING1, regulating H3K9/H3K4 methylation/acetylation, thereby modulating ECM remodeling, apoptosis, and inflammation. Therapeutically, AAV-mediated CSE or CBX3 delivery via extravascular carrier reduced AAD incidence and progression. Thus, VSMC-derived CSE/H₂S\u0026ndash;CBX3 signaling restrains AAD through epigenetic regulation of the ADAMTS4\u0026ndash;versican axis.\u003c/p\u003e","manuscriptTitle":"Chromobox 3 Assembles a Novel Epigenetic Complex Contributing to Cystathionine γ-lyase–mediated Protection Against Aortic Aneurysm/dissection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 06:12:23","doi":"10.21203/rs.3.rs-7423522/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"48eda625-b3a2-402f-8bbb-34c512ff1931","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53989900,"name":"Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases"},{"id":53989901,"name":"Health sciences/Diseases/Cardiovascular diseases"}],"tags":[],"updatedAt":"2026-04-29T00:25:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 06:12:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7423522","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7423522","identity":"rs-7423522","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}