Meta-Analysis of Smooth Muscle Lineage Transcriptomes in Atherosclerosis and Their Relationships to In Vitro Models

Abstract

Background: Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity in atherosclerotic plaques, and among other approaches, has been modeled in vitro by cholesterol loading. Methods: Meta-analysis of scRNA-seq data from VSMC lineage traced cells across five experiments of murine atherosclerosis was performed. In vivo expression profiles were compared to three in vitro datasets of VSMCs loaded with cholesterol and three datasets of polarized macrophages. Results: We identified 24 cell clusters in the meta-analysis of single cells from mouse atherosclerotic lesions with notable heterogeneity across studies, especially for macrophage populations. Trajectory analysis of VSMC lineage positive cells revealed several possible paths of state transitions with one traversing from contractile VSMC to macrophages by way of a proliferative cell cluster. Transcriptome comparisons between in vivo and in vitro states underscored that data from three in vitro cholesterol-treated VSMC experiments did not mirror cell state transitions observed in vivo. However, all in vitro macrophage profiles analyzed (M1, M2, and oxLDL) were more similar to in vivo profiles of macrophages than in vitro VSMCs were to in vivo profiles of VSMCs. oxLDL loaded macrophages showed the most similarity to in vivo states. In contrast to the in vitro data, comparison between mouse and human in vivo data showed many similarities. Conclusions: Identification of the sources of variation across single cell datasets in atherosclerosis will be an important step towards understanding VSMC fate transitions in vivo. Also, we conclude that cholesterol-loading in vitro is insufficient to model the VSMC cell state transitions observed in vivo, which underscores the need to develop better cell models. Mouse models, however, appear to reproduce a number of the features of VSMCs in human plaques.

Mechanical programming of arterial smooth muscle cells in health and ageing

“…Additionally, it would help explain how mechanical insults modulate the transcriptional regulation of ASMC differentiation. These techniques have recently shown that genes associated with actin filament reorganisation become upregulated in ASMCs during ageing or disease but involvement of the mechanical landscape remains unknown (Dobnikar et al 2018;Gao et al 2020;Conklin et al 2021).…”

Mechanisms of vascular smooth muscle cell investment and phenotypic diversification in vascular diseases

“…However, the cell populations referred to as ‘fibromyocytes' [ 24 ], ‘pioneer cells’ [ 20 ] and ‘stem cell, endothelial, monocyte; SEM cells' [ 22 ] share expression of a set of markers including Ly6a /SCA1, Lgals3 and Vcam1 and likely represent similar populations. We henceforth refer to this modulated VSMC state as SEM cells, similar to a recent meta-analysis of this data by Conklin et al [ 30 ]. Modulated VSMC states were also identified in scRNA-seq datasets of human plaques, indicating that they are clinically relevant for human atherogenesis.…”

“…Recent scRNA-seq studies have increased our ability to identify phenotypes adopted by VSMC-derived cell types in vascular disease models beyond staining for a few markers [14,[20][21][22][23][24]48]. Comparison of data from several groups provides robust evidence that VSMC-derived atherosclerotic plaque cells contribute significantly to overlapping cell states comprising fibroblast-like and 'fibrochondrocytic' cells and modulated VSMCs [30,49]. The specific clustering and nomenclature of modulated VSMCs differ between studies.…”

“…Many SCA1+ VSMCs showed significant transcriptional differences to the bulk medial VSMC population, including reduced levels of contractile marker transcripts and expression of activation-associated genes. Notably, an increased proportion of VSMC-derived cells express SCA1+ in the plaque [ 21 , 24 , 30 ], in aneurysm [ 14 ] and after acute injury [ 21 ], which could be accounted for by expansion of an original SCA1+ population. However, definitively proving this using lineage tracing of SCA1+ VSMCs from healthy to diseased conditions is technically challenging as (1) SCA1 is also expressed in adventitial and endothelial cells, necessitating the use of a dual-lineage tracing system [ 20 , 31 ] and (2) SCA1+ VSMCs express the Ly6a transcript at levels likely too low to induce Cre-mediated recombination [ 31 , 32 ].…”

“…Meta-analysis of scRNA-seq datasets generated from VSMC-lineage traced plaques revealed a continuum in transcriptional profiles between contractile and modulated Ly6a+/ SCA1+ ‘SEM' (stem cell, endothelial, monocyte [ 22 ]) cells [ 30 ]. This is inconsistent with a distinct transcriptional response to atherogenic stimuli of a dedicated progenitor population compared with the bulk VSMC population.…”