Aims Vascular smooth muscle cells (SMCs) and their derivatives are key contributors to the development of atherosclerosis. However, studying changes in SMC gene expression in heterogeneous vascular tissues is challenging due to the technical limitations and high cost associated with current approaches. In this paper, we apply Translating Ribosome Affinity Purification sequencing (TRAP-Seq) to profile SMC-specific gene expression directly from tissue. Methods and Results To facilitate SMC-specific translatome analysis, we generated SMCTRAP mice, a transgenic mouse line expressing EGFP-tagged ribosomal protein L10a (EGFP-L10a) under the control of the SMC-specific αSMA promoter. These mice were further crossed with the atherosclerosis model Ldlr-/-, ApoB100/100 to generate SMCTRAP-AS mice and used to profile atherosclerosis-associated SMCs in thoracic aorta samples of 15-month-old SMCTRAP and SMCTRAP-AS mice. Our analysis of SMCTRAP-AS mice showed that EGFP-L10a expression was localized to SMCs in various tissues, including the aortic wall and plaque. The TRAP fraction demonstrated high enrichment of known SMC-specific genes, confirming the specificity of our approach. We identified several genes, including Cemip, Lum, Mfge8, Spp1, and Serpina3, that are known to be involved in atherosclerosis-induced gene expression. Moreover, we identified several novel genes not previously linked to SMCs in atherosclerosis, such as Anxa4, Cd276, Itih4, Myof, Pcdh11x, Rab31, Serpinb6b, Slc35e4, Slc8a3, and Spink5. Among them, we confirmed the SMC-specific expression of Itih4 in atherosclerotic lesions using immunofluorescence staining of mouse aortic roots and spatial transcriptomics of human carotid arteries. Furthermore, our more detailed analysis of Itih4 showed its link to coronary artery disease (CAD) through the colocalization of GWAS, splice-QTL, and protein-QTL signals. Conclusions We generated a SMC-specific TRAP mouse line to study atherosclerosis and identified Itih4 as a novel SMC-expressed gene in atherosclerotic plaques, warranting further investigation of its putative function in extracellular matrix stability and genetic evidence of causality.

中文翻译：

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翻译组分析揭示 Itih4 是动脉粥样硬化中一种新型平滑肌细胞特异性基因

目的 血管平滑肌细胞 (SMC) 及其衍生物是动脉粥样硬化发展的关键因素。然而，由于当前方法的技术限制和高成本，研究异质血管组织中 SMC 基因表达的变化具有挑战性。在本文中，我们应用翻译核糖体亲和纯化测序 (TRAP-Seq) 直接从组织中分析 SMC 特异性基因表达。方法和结果为了促进 SMC 特异性翻译组分析，我们生成了 SMCTRAP 小鼠，这是一种转基因小鼠系，在 SMC 特异性 αSMA 启动子的控制下表达 EGFP 标记的核糖体蛋白 L10a (EGFP-L10a)。这些小鼠进一步与动脉粥样硬化模型 Ldlr-/-、ApoB100/100 杂交，生成 SMCTRAP-AS 小鼠，并用于分析 15 个月大的 SMCTRAP 和 SMCTRAP-AS 小鼠胸主动脉样本中动脉粥样硬化相关的 SMC。我们对 SMCTRAP-AS 小鼠的分析表明，EGFP-L10a 表达局限于各种组织中的 SMC，包括主动脉壁和斑块。 TRAP 部分证明了已知 SMC 特异性基因的高度富集，证实了我们方法的特异性。我们鉴定了几个基因，包括 Cemip、Lum、Mfge8、Spp1 和 Serpina3，这些基因已知参与动脉粥样硬化诱导的基因表达。此外，我们还发现了一些以前未与动脉粥样硬化中的 SMC 相关的新基因，例如 Anxa4、Cd276、Itih4、Myof、Pcdh11x、Rab31、Serpinb6b、Slc35e4、Slc8a3 和 Spink5。其中，我们通过小鼠主动脉根部的免疫荧光染色和人颈动脉的空间转录组学证实了 Itih4 在动脉粥样硬化病变中的 SMC 特异性表达。此外，我们对 Itih4 的更详细分析表明，它通过 GWAS、剪接 QTL 和蛋白质 QTL 信号的共定位与冠状动脉疾病 (CAD) 存在关联。结论 我们构建了 SMC 特异性 TRAP 小鼠系来研究动脉粥样硬化，并将 Itih4 鉴定为动脉粥样硬化斑块中新型 SMC 表达基因，值得进一步研究其在细胞外基质稳定性中的推定功能和因果关系的遗传证据。