Single-Molecule Spatial Transcriptomics of Human Thoracic Aortic Aneurysms Uncovers Calcification-Related CARTPT -Expressing Smooth Muscle Cells

Abstract

Background: Although single-cell RNA-sequencing is commonly applied to dissect the heterogeneity in human tissues, it involves the preparation of single-cell suspensions via cell dissociation, causing loss of spatial information. In this study, we employed high-resolution single-cell transcriptome imaging to reveal rare smooth muscle cell (SMC) types in human thoracic aortic aneurysm (TAA) tissue samples. Methods: Single-molecule spatial distribution of transcripts from 140 genes was analyzed in fresh-frozen human TAA samples with region and sex-matched controls. In vitro studies and tissue staining were performed to examine human CART prepropeptide ( CARTPT ) regulation and function. Results: We captured thousands of cells per sample including a spatially distinct CARTPT -expressing SMC subtype enriched in male TAA samples. Immunoassays confirmed human CART enrichment in male TAA tissue and truncated CARTPT secretion into cell culture medium. Oxidized low-density lipoprotein, a cardiovascular risk factor, induced CARTPT expression, whereas CARTPT overexpression in human aortic SMCs increased the expression of key osteochondrogenic transcription factors and reduced contractile gene expression. Recombinant human CART treatment of human SMCs further confirmed this phenotype. Alizarin red staining revealed calcium deposition in male TAA samples showing similar localization with human CART staining. Conclusions: Here, we demonstrate the feasibility of single-molecule imaging in uncovering rare SMC subtypes in the diseased human aorta, a difficult tissue to dissociate. We identified a spatially distinct CARTPT -expressing SMC subtype enriched in male human TAA samples. Our functional studies suggest that human CART promotes osteochondrogenic switch of aortic SMCs, potentially leading to medial calcification of the thoracic aorta.

SWI/SNF Complex in Vascular Smooth Muscle Cells and Its Implications in Cardiovascular Pathologies

“…Vascular smooth muscle cell plasticity in CVD, including atherosclerosis, has been well-documented [2]. Especially in the past decade, the emergence of new technologies, such as lineage tracing, single-cell RNA sequencing (scRNA-Seq), and spatial transcriptomics [18][19][20][21], has largely extended our knowledge of cell origin, characteristics, and transcriptomic profiles in the atherosclerotic lesions. Under pathological conditions, VSMCs transdifferentiate into various cell types such as foam cells [22,23], mesenchymal-stem-cell (MSC)-like cells [24], macrophage-like cells [25,26], adipocyte-like cells [27], osteochondrogenic cells [28,29], fibromyocytes [19].…”

Discovering a Rare Smooth Muscle Cell Population Specific to Men in Ascending Aortic Aneurysm Using Spatial Transcriptomics

“…In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, a groundbreaking study by Mizrak et al 11 meets the challenge of identifying aortic cellular transcriptomes while preserving spatial resolution. The authors use multiplexed error-robust fluorescence in situ hybridization, a cutting-edge in situ hybridization technique that couples single-molecule, single-cell transcriptomics with high-resolution spatial imaging, to obtain a more comprehensive view of the molecular basis of ascending aortic aneurysms.…”

Editorial: Single-cell OMICs analyses in cardiovascular diseases

“…Marrero et al study on sexual differences in peripheral artery disease (PAD) etiology utilizing scRNA-seq data. Mizrak et al ( 12 ) using spatial transcriptomics uncovered male-specific smooth muscle cells subpopulation playing a key role in human thoracic aortic aneurysm. ScRNA-seq data from Shin et al ( 13 ) allow to delineate the sex-difference in endothelial cells characteristics and function providing new clues about atherosclerotic diseases.…”