研究背景和目的：

腹主动脉瘤（Abdominal aortic aneurysm, AAA）是指腹主动脉局部永久性扩张，超过正常血管直径50%或腹主动脉的直径大于3cm。作为一种潜在的致命性血管疾病，AAA病理生理过程主要包括内皮细胞粘附分子和趋化因子表达增加、血管平滑肌细胞（Vascular smooth muscle cells, VSMCs） 减少和功能障碍、主动脉壁炎症、血管细胞外基质降解、重塑等。关于AAA的治疗方法，除开放手术或血管内修复术外，尚缺乏有效的药物干预方法延缓AAA的进展。

硫化氢（Hydrogen sulfide, H₂S）作为一种新的具有心血管保护效应的气体信号分子，在体内主要依赖以L-半胱氨酸或L-同型半胱氨酸为底物，依赖胱硫醚β合酶、胱硫醚γ裂解酶（cystathionine γ lyase, CSE）、以及β-巯基丙酮酸转硫酶催化合成。在心血管组织中，主要表达CSE，而β-巯基丙酮酸转硫酶的底物为L-半胱氨酸的代谢产物—β巯基丙酮酸，因此，CSE是心血管组织及细胞内源性产生H₂S的关键酶。在主动脉夹层及主动脉瘤病人样品中，应用蛋白免疫印迹法（Western Blot，WB）检测CSE蛋白表达显著下调。随着年龄增加，血管组织中CSE表达显著下调；而全身敲除CSE，随着年龄增加，主动脉直径显著高于对照小鼠，且在血管紧张素II（Angiotensin II, AngII）诱导下主动脉扩张更加显著。机制研究也发现，CSE/H₂S可以诱导基质金属蛋白酶2和9（Matrix Metalloprotease2/9, MMP2/9）的S-sulfhydration修饰而抑制MMP2和MMP9的活性。而内皮细胞特异性敲除CSE，可通过减少蛋白质二硫键异构酶的S-sulfhydration，促进内皮细胞的内质网应激进而促进主动脉夹层和动脉瘤的发展。既往研究和我们的前期工作发现，动脉组织中CSE主要表达于平滑肌细胞，那么，平滑肌细胞CSE/H₂S在主动脉瘤发生发展中的病理生理调节和意义尚未明确。

我们采用高通量测序分析（GEO number: GSE290627）分析结合基因重要性联合分析发现，CSE敲除的VSMCs在AngII刺激后，神经肿瘤腹侧抗原2（neuro-oncology ventral antigen 2, NOVA2）表达显著下调。NOVA2作为一种RNA剪接调节因子参与血管发育和管腔形成。RNA剪接是真核生物控制基因表达的一种十分重要的机制，其中选择性剪接使生物体从单个转录本剪切生成多个RNA，进而翻译多个序列同源但功能不同的蛋白质。研究表明各种剪接因子调控的选择性剪接（如VEGF的不同剪切体），与心脏发育、血管新生、动脉粥样硬化等各种心血管疾病的病理生理学密切相关。因此，根据文献报道及我们的前期工作，我们提出“VSMC内源性的CSE/H₂S可能通过NOVA2，调节VSMCs功能，参与腹主动脉瘤的发生发展”的科学假说。本研究应用模式动物及分子生物学手段，旨在证明这一假说。

研究方法：

（1）通过生物信息学的方法分析AngII诱导下CSE-KO细胞差异表达基因，以及RIP-seq和CircRNA测序分析NOVA2调节的下游靶点；（2）AAA动物模型：构建平滑肌细胞CSE特异性基因敲除小鼠（CSE^SMCKO）和过表达小鼠（CSE^SMCKI），通过腺相关病毒腹主动脉局部过表达NOVA2或局部敲除NOVA2，并使用AngII（1000 ng/kg/min）诱导28天构成AAA模型；（3）细胞模型：WT和CSE-KO小鼠主动脉平滑肌细胞，通过转染siRNA或腺病毒实现敲低NOVA2和过表达NOVA2，并给予AngII（1μM）刺激24h，模拟体内腹主动脉瘤环境；（4）通过超声心动图检测小鼠腹主动脉成瘤情况，腹主动脉病理实验观察小鼠腹主动脉病变情况；（5）通过免疫共沉淀和定量实时聚合酶链式反应（Quantitative Reverse Transcription Polymerase Chain Reaction, qRT-PCR）、免疫荧光染色观察平滑肌细胞中NOVA2表达情况；（6）EDU染色、Transwell/wound healing划痕实验、Tunel染色/ Annexin V-FITC细胞凋亡流式分别检测细胞增殖、迁移和凋亡情况；（7）通过RNA免疫共沉淀测序（RNA immunoprecipitation sequencing, RIP-seq），分析NOVA2的靶基因并验证其介导的VSMC功能。

研究结果：

AngII刺激WT和CSE敲除（Knockout, KO）小鼠的平滑肌细胞，24h后qRT-PCR确证CSE敲除的VSMCs在AngII诱导下NOVA2 mRNA表达显著下调（与RNA-seq结果一致），Western blot发现NOVA2蛋白表达在CSE-KO细胞表达也显著下调。免疫荧光染色发现，AAA病人的样本，以及AngII诱导的BAPN和PCSK9的AAA模型的主动脉瘤体中NOVA2表达也显著下调。

应用LoxP-Cre重组酶技术制备了平滑肌细胞特异性敲除CSE（CSE^SMCKO）以及平滑肌细胞特异性敲入（CSE^SMCKI）小鼠。尾静脉注射AAV8-ApoEHCR-hAAT-D377Y-mPCSK9腺相关病毒在肝脏过表达前蛋白转化酶枯草溶菌素9（Proprotein Convertase Subtilisin/Kexin Type 9, PCSK9），并予以高脂饮食制备高脂血症；或者予以β-氨基丙腈（β-aminopropionitrile, BAPN）饮水，抑制赖氨酰氧化酶活性，干扰弹性蛋白和胶原纤维的交联。在此基础上，予AngII（1000 ng/kg/min）缓释泵输注4周制备AAA小鼠模型。与对照组（CSE^LoxP）小鼠相比，CSE^SMCKO小鼠的AAA的发病率增加，腹主动脉扩张程度加重。而在CSE^SMCKO小鼠腹主动脉使用NOVA2腺相关病毒（AAV9-SM22α-ΝΟVA2）局部过表达NOVA2后，与对照病毒（AAV9-SM22α-GFP）相比，小鼠AAA发病率显著降低，同时也缓解了血管扩张、弹力纤维断裂和胶原蛋白降解的程度。反之，CSE^SMCKI小鼠腹主动脉应用AAV9-shNOVA2腺相关病毒局部敲低NOVA2后，与对照病毒（腹主动脉局部感染插入无义序列的AAV9-shscramble）相比，BAPN+AngII诱导的AAA发生率显著增加。H&E、EVG和Masson染色结果显示，敲低NOVA2后，小鼠血管扩张、弹力纤维断裂、胶原降解加重。以上功能获得性/缺失性研究证实，平滑肌细胞内源性CSE/H₂S通过NOVA2参与AAA的发生发展。

原代培养野生型小鼠和CSE敲除小鼠的主动脉平滑肌细胞，予AngII刺激，CSE-KO平滑肌细胞增殖能力和迁移能力降低，细胞凋亡增加。过表达NOVA2腺病毒，挽救了CSE-KO VSMCs细胞的增殖和迁移，凋亡减少。用siRNA敲低NOVA2后，CSE-KO平滑肌细胞增殖能力和迁移能力进一步降低，细胞凋亡显著增加。通过NOVA2的RIP-seq分析，发现NOVA2可能结合的RNA 969个，基因功能分析富集在细胞外基质，凋亡细胞清除，血压调节，RNA结合等通路。RNA-seq分析，RNA的可变剪切主要发生外显子跳跃56.92 %。联合分析发现，CSE/H₂S通过NOVA2参与RNA可变剪切的靶基因133个，功能主要富集于细胞外基质，凋亡细胞清除，血压调节，RNA结合等信号通路。这些基因在NOVA2作用下如何剪切，其在平滑肌细胞功能和AAA发病中的病理生理调节尚需下一步工作进行深入探讨。

研究结论：

本研究证实，平滑肌细胞内源性CSE/H₂S可以通过NOVA2，调节平滑肌细胞的增殖迁移及凋亡，参与腹主动脉瘤形成。本研究阐明了平滑肌细胞内源性CSE/H₂S作用于AAA的新的调控机制，也丰富了NOVA2在血管平滑肌细胞中的功能及其靶基因选择性剪接介导的病理生理新机制。

Background and Objectives：

Abdominal aortic aneurysm (AAA) refers to a localized, permanent dilation of the abdominal aorta that exceeds 50% of the normal vessel diameter or has an absolute diameter greater than 3 cm. As a potentially fatal vascular disease, the pathophysiological process of AAA primarily includes increased expression of endothelial adhesion molecules and chemokines, vascular smooth muscle cell (VSMC) reduction and dysfunction, aortic wall inflammation, and vascular extracellular matrix degradation and remodeling. Regarding AAA treatment, apart from open surgery or endovascular repair, there are currently no effective pharmacological interventions to delay AAA progression.

Hydrogen sulfide (H₂S), a newly recognized gaseous signaling molecule with cardiovascular protective effects, is endogenously synthesized from L-cysteine or L-homocysteine by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). In cardiovascular tissues, CSE is predominantly expressed, while 3-MST utilizes β-mercaptopyruvate, a metabolic product of L-cysteine, as its substrate. Therefore, CSE is the key enzyme responsible for the endogenous production of H₂S in cardiovascular tissues and cells. Western blot analysis of samples from patients with aortic dissection and aortic aneurysms has shown a significant downregulation of CSE protein expression. With aging, CSE expression in vascular tissues declines significantly. Additionally, systemic CSE knockout mice exhibit a greater aortic diameter with age, and their aortic dilation is further exacerbated under angiotensin II (Ang II) induction.

Mechanistic studies have shown that the CSE/H₂S system induces the S-sulfhydration modification of matrix metalloproteinases 2 and 9 (MMP2/9), thereby inhibiting their activity. Furthermore, endothelial cell-specific CSE knockout can reduce S-sulfhydration of protein disulfide isomerase (PDI), leading to increased endoplasmic reticulum (ER) stress in endothelial cells and thereby promoting the development of aortic dissection and aneurysms.

Previous studies and our preliminary work indicate that CSE in vascular tissues is primarily expressed in smooth muscle cells. However, the pathophysiological role and significance of smooth muscle cell-specific CSE/H₂S in AAA development remain unclear.

Using high-throughput sequencing analysis (GEO number: GSE290627), we identified that NOVA2 (Neuro-Oncology Ventral Antigen 2) expression was significantly downregulated in CSE-knockout (CSE-KO) VSMCs stimulated with Ang II. NOVA2, as an RNA splicing regulator, is involved in vascular development and lumen formation. RNA splicing is a crucial gene expression regulatory mechanism in eukaryotic cells, where alternative splicing generates multiple RNA isoforms from a single transcript, leading to the translation of proteins with different functions. Studies have shown that alternative splicing events regulated by various splicing factors, such as VEGF isoforms, are closely associated with the pathophysiology of cardiovascular diseases, including cardiac development, angiogenesis, and atherosclerosis.

Based on previous studies and our preliminary findings, we propose the hypothesis that endogenous CSE/H₂S in VSMCs may regulate NOVA2, thereby modulating VSMC function and participating in AAA development. This study employs animal models and molecular biology approaches to verify this hypothesis.

Research Methods：

(1) Use bioinformatics methods to analyze differentially expressed genes in CSE-KO cells induced by Ang II, as well as RIP-seq and CircRNA sequencing to analyze the downstream targets regulated by NOVA2; (2) AAA animal model: Construct smooth muscle cell-specific CSE knockout mice (CSE^SMCKO) and overexpression mice (CSE^SMCKI), locally overexpress or knock down NOVA2 in the abdominal aorta using adeno-associated virus (AAV), and induce the AAA model with Ang II (1000 ng/kg/min) for 28 days; (3) Cell model: Primary aortic smooth muscle cells (VSMCs) from WT and CSE-KO mice are transfected with siRNA or adenovirus to knock down or overexpress NOVA2, followed by stimulation with Ang II (1μM) for 24 hours to simulate the in vivo AAA environment; (4） Use echocardiography to assess aneurysm formation in the abdominal aorta of mice and perform histopathological experiments to examine the vascular pathology of the abdominal aorta; (5) Detect NOVA2 expression in smooth muscle cells using co-immunoprecipitation, qRT-PCR, and immunofluorescence staining; (6) Perform EDU staining, Transwell/wound healing assays, and Tunel staining/Annexin V-FITC apoptosis flow cytometry to evaluate cell proliferation, migration, and apoptosis; (7) Conduct RNA immunoprecipitation sequencing (RIP-seq) to analyze NOVA2 target genes and verify its role in regulating VSMC function.

Results：

After 24 hours of AngII stimulation in wild-type (WT) and CSE knockout (KO) VSMCs, qRT-PCR confirmed that NOVA2 mRNA expression was significantly downregulated in CSE-KO VSMCs under AngII induction, consistent with RNA-seq results. Western blot analysis further revealed a significant reduction in NOVA2 protein expression in CSE-KO cells. Immunofluorescence staining demonstrated that NOVA2 expression was also markedly downregulated in aortic aneurysm tissues from AAA patients, as well as in the aneurysmal tissues of the AngII-induced BAPN and PCSK9 AAA models.

Using the LoxP-Cre recombinase system, we generated smooth muscle cell-specific CSE knockout (CSE^SMCKO) and smooth muscle cell-specific CSE knock-in (CSE^SMCKI) mice. To induce hyperlipidemia, AAV8-ApoEHCR-hAAT-D377Y-mPCSK9 adeno-associated virus was administered via tail vein injection to overexpress proprotein convertase subtilisin/kexin type 9 (PCSK9) in the liver, followed by a high-fat diet. Alternatively, β-aminopropionitrile (BAPN) was administered via drinking water to inhibit lysyl oxidase activity, disrupting elastin and collagen fiber cross-linking. Based on these conditions, an AAA mouse model was established by continuous Ang II (1000 ng/kg/min) infusion via osmotic pump for four weeks. Compared to control (CSE^LoxP) mice, CSE^SMCKO mice exhibited an increased incidence of AAA and greater abdominal aortic dilation.

In CSE^SMCKO mice, local overexpression of NOVA2 in the abdominal aorta using NOVA2 adeno-associated virus (AAV9-SM22α-NOVA2) significantly reduced AAA incidence compared to the control virus (AAV9-SM22α-GFP). This intervention also alleviated vascular dilation, elastic fiber fragmentation, and collagen degradation. Conversely, in CSE^SMCKI mice, local knockdown of NOVA2 in the abdominal aorta using AAV9-shNOVA2 significantly increased the incidence of BAPN+AngII-induced AAA compared to the control virus (AAV9-shScramble, carrying a nonsense sequence). H&E, EVG, and Masson staining further demonstrated that NOVA2 knockdown exacerbated vascular dilation, elastic fiber rupture, and collagen degradation. These gain-of-function and loss-of-function studies confirmed that smooth muscle cell-endogenous CSE/H₂S contributes to AAA development via NOVA2.

Primary aortic smooth muscle cells from WT and CSE-KO mice were stimulated with Ang II. CSE-KO VSMCs exhibited reduced proliferation and migration but increased apoptosis. Overexpression of NOVA2 via adenoviral transduction rescued the proliferation and migration capacity of CSE-KO VSMCs while reducing apoptosis. Conversely, siRNA-mediated knockdown of NOVA2 further decreased proliferation and migration while significantly increasing apoptosis in CSE-KO VSMCs. Primary aortic smooth muscle cells (VSMCs) were cultured from wild-type and CSE knockout (CSE-KO) mice and stimulated with AngII. CSE-KO VSMCs exhibited reduced proliferation and migration capacity, along with increased apoptosis. Overexpression of NOVA2 via adenoviral transduction rescued the proliferation and migration abilities of CSE-KO VSMCs and reduced apoptosis. Conversely, siRNA-mediated knockdown of NOVA2 further impaired proliferation and migration while significantly increasing apoptosis in CSE-KO VSMCs.

RIP-seq analysis of NOVA2 identified 969 potential RNA-binding targets, which were functionally enriched in pathways related to extracellular matrix organization, apoptotic cell clearance, blood pressure regulation, and RNA binding. RNA-seq analysis revealed that alternative splicing events in CSE-KO VSMCs primarily involved exon skipping (56.92%). Integrated analysis identified 133 NOVA2-regulated alternative splicing target genes modulated by the CSE/H₂S pathway, with functions enriched in extracellular matrix organization, apoptotic cell clearance, blood pressure regulation, and RNA binding pathways. Further research is needed to elucidate how NOVA2-mediated alternative splicing regulates VSMC function and contributes to the pathophysiology of AAA.

Conclusions：

This study demonstrates that endogenous CSE/H₂S in VSMCs regulates NOVA2, influencing VSMC proliferation, migration, and apoptosis, thereby contributing to AAA formation. Our findings reveal a novel regulatory mechanism involving CSE/H₂S and NOVA2 in AAA pathogenesis. Additionally, this study expands our understanding of NOVA2 function in vascular smooth muscle cells and its role in alternative splicing-mediated vascular pathology.