The applications of single-cell RNA sequencing in atherosclerotic research

Abstract

At the base of the work described here is the overview of all different cell populations present in the carotid atherosclerotic plaque, which were previously not characterized in such detail. We unraveled cell populations such as smooth muscle cells (SMCs), endothelial cells (ECs) and various immune cells via single cell RNA-sequencing (scRNA-seq) which were characterized by examining their specific marker genes. We examined potential ligand-receptor interactions to study cell-cell communications within the plaque and found that myeloid, EC and SMCs are most likely to be interacting. In addition, we compared the human myeloid populations with mouse populations and reported a decent concordance in cell type diversity between species.

We leveraged this new knowledge of the cell populations to study the wealth of genome wide association studies (GWAS) loci associated with cardiovascular disease. Identifying the genes from GWAS that are potential candidate genes for clinical care is not straightforward. We describe a two-step approach how we can use scRNA-seq data to translate GWAS susceptibility loci into targets for clinical care by directly examining expression in disease relevant tissue. Loci associated with coronary artery disease are dominantly associated with plaque SMC and EC populations. After identifying (novel) cell- gene pairs, expression of SKI, KANK2 and EDNRA was correlated with migration, proliferation and calcification in vascular SMCs.

The molecular mechanisms of endothelial to mesenchymal transition (EndoMT) in human atherosclerosis are poorly understood. We combined the power of in vitro experimental data and scRNA-seq of a lineage tracing mouse model to help study EndoMT in human lesions. We assessed the temporal gene expression under EndoMT promoting conditions and defined assorted patterns of gene expression over the course of time. Next we studied scRNA-seq data from a lineage tracing mouse model were we composed endoMT trajectories. This data was used to demonstrate that the temporal gene expression changes from the in vitro model could identify EndoMT trajectories. Finally, we constructed three candidate EndoMT lineages across multiple subpopulations of ECs and SMCs in human carotid scRNA-seq data, similar to the approach that was applied in mouse data. We examined gene expression over the course of these lineages and identified 73 potential markers for mid-stage EndoMT. From these genes, NRG1 and DEPP1 are associated with mid-stage EndoMT in human atherosclerotic cells.

Lastly we studied sex differences in human atherosclerosis via (sc)RNA-seq data. We compared SMC and EC sex-stratified gene expression profiles from 20 female and 26 male atherosclerotic lesions. And used deconvolution to estimate cell population proportions of RNA seq from 169 female and 485 male samples. Differences in sex-related biological pathways in sex stratified scRNA-seq data revealed that the differences in genes found between sexes do not have a common denominator such as “apoptosis” or “endoMT”, but could individually point to different processes. Deconvolution results indicated that female plaques showed a trend towards a higher SMC content compared to males and that males showed a trend towards a higher macrophage and immune cell content. Consequently, females leaned towards a higher ratio of structural vs. immune cells.