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Background:Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation‚ notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies.Methods:We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity.Results:In addition to macrophages, we found a high proportion of αβ T cells in the coronary plaques. Most of these T cells lack high expression ofCCR7andL-selectin, indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express theHLA-DRAsurface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αβ T cells (CD4HLA-DRA, 2 clusters expressed a proinflammatory and cytolytic signature characteristic of CD8 cells, while the other expressed AREG (amphiregulin), which promotes smooth muscle cell proliferation and fibrosis, and, thus, contributes to plaque progression.Conclusions:Taken together, these findings demonstrate that plaque T cells are clonally expanded potentially by antigen engagement, are potentially reactive to self-epitopes, and may interact with smooth muscle cells and macrophages in the plaque microenvironment.

Rationale: Atherosclerosis is characterized by an accumulation of foam cells within the arterial wall, resulting from excess cholesterol uptake and buildup of cytosolic lipid droplets (LDs). Autophagy promotes LD clearance by freeing stored cholesterol for efflux, a process that has been shown to be atheroprotective. While the role of autophagy in LD catabolism has been studied in macrophage-derived foam cells, this has remained unexplored in vascular smooth muscle cell (VSMC)-derived foam cells that constitute a large fraction of foam cells within atherosclerotic lesions. Objective: We performed a comparative analysis of autophagy flux in lipid-rich aortic intimal populations to determine whether VSMC-derived foam cells metabolize LDs similarly to their macrophage counterparts. Methods and Results: Atherosclerosis was induced in GFP-LC3 (microtubule-associated proteins 1A/1B light chain 3) transgenic mice by PCSK9 (proprotein convertase subtilisin/kexin type 9)-adeno-associated viral injection and Western diet feeding. Using flow cytometry of aortic digests, we observed a significant increase in dysfunctional autophagy of VSMC-derived foam cells during atherogenesis relative to macrophage-derived foam cells. Using cell culture models of lipid-loaded VSMCs and macrophages, we show that autophagy-mediated cholesterol efflux from VSMC foam cells was poor relative to macrophage foam cells, and largely occurs when HDL (high-density lipoprotein) was used as a cholesterol acceptor, as opposed to apoA-1 (apolipoproteinA-1). This was associated with the predominant expression of ABCG1 in VSMC foam cells. Using metformin, an autophagy activator, cholesterol efflux to HDL was significantly increased in VSMC, but not in macrophage, foam cells. Conclusions: These data demonstrate that VSMC and macrophage foam cells perform cholesterol efflux by distinct mechanisms, and that autophagy flux is highly impaired in VSMC foam cells, but can be induced by pharmacological means. Further investigation is warranted into targeting autophagy specifically in VSMC foam cells, the predominant foam cell subtype of advanced atherosclerotic plaques, to promote reverse cholesterol transport and resolution of the atherosclerotic plaque.

Background: Human vascular diseases are the worldwide leading causes of morbidity and mortality. Nearly all human vascular diseases have arterial segment-specific tropisms despite identical exposures to genetic and environmental risk factors. Understanding the cellular and transcriptomic determinants of arterial identities may hold the key to identifying novel pathophysiology and potential therapies. Methods: To specifically determine arterial site-specific differences independent of inter-individual variation, we have generated a human arterial cellular atlas by simultaneously collecting and analyzing up to 8 arterial sites from multiple healthy transplant donors. We performed single cell transcriptomic analysis on arterial segments to determine the differences in cellular composition and transcriptomic programs. We subsequently integrated human genetic data with cell-type specific transcriptomic differences across vascular beds to identify probable causal cells and causal genes associated with human vascular phenotypes. Results/Conclusions: Single cell transcriptomic analysis of >150,000 cells sequenced at >50,000 reads per cell revealed that the dominant cellular drivers of transcriptomic differences between distinct arterial segments, i.e. determinants of arterial identity, are fibroblasts and smooth muscle cells, not endothelial cells or macrophages. Adult vascular cells transcriptomes from different segments are most influenced by their embryonic origins but not by anatomical proximity. Differentially regulated genes in fibroblast across different vascular beds were particularly enriched for vascular disease associated genetic signals, suggesting a prominent role for these cells in human disease. While the majority of endothelial cells were transcriptionally similar across vascular beds, a rare, previously undescribed, cluster of endothelial cells were identified who expressed segment-specific transcriptomic signatures. Differentially expressed genes in these cells were enriched for vascular disease signals, suggesting a possible role of these rare cells in human disease.