Atherosclerotic lesions are known for their complex and diverse cellular heterogeneity, yet the exact cellular composition of human plaques remains unclear. Here we aim to provide a comprehensive... Show moreAtherosclerotic lesions are known for their complex and diverse cellular heterogeneity, yet the exact cellular composition of human plaques remains unclear. Here we aim to provide a comprehensive overview of the cellular content of advanced atherosclerotic lesions using single-cell RNA sequencing (scRNAseq), to increase our understanding of the pathophysiologic processes underlying atherosclerosis.Advanced carotid plaques were obtained from 3 male endarterectomy patients. Plaques were enzymatically digested and live cells were sorted using FACS. Subsequently, we performed scRNAseq on a total of 3456 plaque derived cells.Unsupervised clustering revealed 11 distinct cell populations, including macrophages, CD4+and CD8+T-cells, B-cells, mast cells, endothelial cells, and smooth muscle cells. Interestingly, we were able to distinguish multiple subclusters for the smooth muscle cells, macrophages, and T-cells. We observed a contractile and synthetic smooth muscle cell cluster and three different macrophage clusters, including inflammatory, wound healing and antigen presenting macrophages. Finally, CD8+T-cells could be subdivided into three clusters based on their activation state, with a clear division between cytolytic effectors and quiescent memory CD8+T-cells. We developed a technique to perform scRNAseq on advanced human atherosclerotic lesions to unravel the cellular landscape within the plaques. We now provide, for the first time, an explicit expression profile of the different cells and their subtypes in atherosclerosis. Show less
Laan, S.W. van der; Slenders, L.; Depuydt, M.; Prange, K.; Granneman, L.; Elbersen, D.; ... ; Pasterkamp, G. 2019
To date, genome-wide association studies (GWAS) have identified hundreds of risk loci for coronary artery disease (CAD), and other cardiometabolic diseases and traits. However, identifying the key... Show moreTo date, genome-wide association studies (GWAS) have identified hundreds of risk loci for coronary artery disease (CAD), and other cardiometabolic diseases and traits. However, identifying the key genes for atherosclerotic disease in these loci remains challenging. Here, we systematically mapped 14 GWAS and leveraged transcriptomics of advanced atherosclerotic plaques (AP) at a single-cell resolution.We isolated viable, nucleated single-cells from plaques of 3 carotid endarterectomy patients using enzymatic digestion and fluorescence-activated cell sorting. We applied a CEL-seq2/SORT-seq protocol and the Seurat pipeline for single-cell RNA sequencing (scRNAseq) and cell identification, respectively. Next we annotated public GWAS data of cardiovascular diseases, and cardiometabolic traits using FUMA, which is based on LD clumping, physical location, regulatory and transcriptomic data.Using scRNAseq we identified 11 cellular clusters in AP, and integrated these data to map 1,336 loci across 14 cardiometabolic GWAS. For CAD 105 mapped genes in 35 established loci were differentially expressed between cellular clusters. Some of these loci harboured upto 10 differentially expressed genes, highly expressed in endothelial cells, mast cells, and smooth muscle cells. Notably, some CAD genes are almost exclusively expressed in a specific cell: in the NOS3 locus, KCNH2 is highly expressed in mast cells, whereas NOS3 itself, but also AMPD2 (SORT1 locus) are highly expressed in endothelial cells.We systematically mapped and annotated risk loci, and integrated this at a single-cell resolution with transcriptomics from AP. We identified specific genes and cellular clusters relevant to atherosclerotic plaques development and progression, informative for future mechanistic studies. Show less
Duijn, J. van; Elsas, M. van; Benne, N.; Depuydt, M.; Wezel, A.; Smeets, H.; ... ; Slutter, B. 2019
CD8+ T-cells can be atheroprotective in clinically relevant advanced stages of atherosclerosis, as their depletion results in less stable lesions with a more inflammatory phenotype. However, the... Show moreCD8+ T-cells can be atheroprotective in clinically relevant advanced stages of atherosclerosis, as their depletion results in less stable lesions with a more inflammatory phenotype. However, the phenotype and function of these cells in the lesional microenvironment remains to be determined. Here, we address how the atherosclerotic environment affects the functionality of CD8+ T-cells.We compared the cytokine production of CD8+ T-cells derived from spleens and aortas of apoE-/- mice with advanced atherosclerosis by flow cytometry.CD8+ T-cells isolated from atherosclerotic lesions produced lower amounts of IFN-γ and TNF-α than their splenic counterparts. The observed dysfunctional phenotype of the lesion-derived CD8+ T-cells was associated with an increased expression of the ectonucleotidase CD39, which converts inflammatory extracellular ATP into immunomodulatory adenosine. Indeed, pharmacological inhibition of CD39 in apoE-/- mice partly restored cytokine production by CD8+ T-cells. Using a bone-marrow transplantation approach, we showed that induction of CD39 was a consequence of antigen-specific CD8+ T-cell activation via T-cell receptor (TCR) signaling within the lesions. Importantly, analysis of human endarterectomy samples showed a clear microenvironment specific upregulation of CD39 on CD8+ T-cells in the plaques of human patients compared to matched CD8+ T-cells from the blood .Our results indicate that the continuous TCR signaling in the atherosclerotic plaque induces an immune regulatory CD8+ T-cell phenotype that is associated with decreased cytokine production through increased CD39 expression in both a murine atherosclerotic model and in atherosclerosis patients. This provides a new understanding of atheroprotective immune regulation by CD8+ T-cells. Show less
Duijn, J. van; Elsas, M. van; Benne, N.; Depuydt, M.; Wezel, A.; Smeets, H.; ... ; Slütter, B. 2019
CD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs.... Show moreCD8+T-cells have been attributed both atherogenic and atheroprotective properties, butanalysis of CD8+T-cells has mostly been restricted to the circulation and secondary lymphoid organs. Theatherosclerotic lesion, however, is a complex microenvironment containing a plethora of inflammatory signals,which may affect CD8+T-cell activation. Here, we address how this environment affects the functionality ofCD8+T-cells. We compared the cytokine production of CD8+T-cells derived from spleens and en-zymatically digested aortas ofapoE−/−mice with advanced atherosclerosis byflow cytometry. Aortic CD8+T-cells produced decreased amounts of IFN-γand TNF-αcompared to their systemic counterparts. The observeddysfunctional phenotype of the lesion-derived CD8+T-cells was not associated with classical exhaustion mar-kers, but with increased expression of the ectonucleotidase CD39. Indeed, pharmacological inhibition of CD39 inapoE−/−mice partly restored cytokine production by CD8+T-cells. Using a bone-marrow transplantation ap-proach, we show that TCR signaling is required to induce CD39 expression on CD8+T-cells in atheroscleroticlesions. Importantly, analysis of human endarterectomy samples showed a strong microenvironment specificupregulation of CD39 on CD8+T-cells in the plaques of human patients compared to matched blood samples. Our results suggest that the continuous TCR signaling in the atherosclerotic environment in thevessel wall induces an immune regulatory CD8+T-cell phenotype that is associated with decreased cytokineproduction through increased CD39 expression in both a murine atherosclerotic model and in atherosclerosispatients. This provides a new understanding of immune regulation by CD8+T-cells in atherosclerosis. Show less
