Translatome profiling reveals Itih4 as a novel smooth muscle cell-specific gene in atherosclerosis

Aarthi Ravindran; Lari Holappa; Henri Niskanen; Ilya Skovorodkin; Susanna Kaisto; Mustafa Beter; Miika Kiema; Ilakya Selvarajan; Valtteri Nurminen; Einari Aavik; Rédouane Aherrahrou; Sanna Pasonen-Seppänen; Vittorio Fortino; Johanna P Laakkonen; Seppo Ylä-Herttuala; Seppo Vainio; Tiit Örd; Minna U Kaikkonen

doi:10.1093/cvr/cvae028

Translatome profiling reveals Itih4 as a novel smooth muscle cell-specific gene in atherosclerosis

Cardiovasc Res. 2024 Jan 30:cvae028. doi: 10.1093/cvr/cvae028. Online ahead of print.

Authors

Aarthi Ravindran¹, Lari Holappa¹, Henri Niskanen¹, Ilya Skovorodkin², Susanna Kaisto², Mustafa Beter¹, Miika Kiema¹, Ilakya Selvarajan¹, Valtteri Nurminen¹, Einari Aavik¹, Rédouane Aherrahrou^{1

3}, Sanna Pasonen-Seppänen⁴, Vittorio Fortino⁴, Johanna P Laakkonen¹, Seppo Ylä-Herttuala¹, Seppo Vainio², Tiit Örd¹, Minna U Kaikkonen¹

Affiliations

¹ A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland.
² Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu.
³ Institute for Cardiogenetics, Universität zu Lübeck; DZHK (German Centre for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck, Germany; University Heart Centre Lübeck, 23562 Lübeck, Germany.
⁴ Institute of Biomedicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland, 70211, Kuopio, Finland.

PMID: 38289873
DOI: 10.1093/cvr/cvae028

Abstract

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.

Keywords: Itih4; Mouse models; RPL10a; Smooth muscle cells; TRAP sequencing; atherosclerosis.