Search

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.

Atherosclerosis is characterized by a build-up of foam cells in the arterial wall, resulting from excess cholesterol uptake and accumulation of cytosolic lipid droplets (LDs). Autophagy has been shown to be atheroprotective in part by promoting the catabolism of LDs which liberates free cholesterol for efflux out of foam cells to cholesterol acceptors (ApoA-I or HDL) for removal from the body. Apart from macrophages (MΦ), vascular smooth muscle cells (VSMCs) comprise 50-70% of foam cells in the plaques. Unlike MΦ, the capacity of VSMC foam cells to metabolize cholesterol via autophagy is unknown. Here, we performed a comparative analysis of the autophagic capacity and cholesterol efflux of arterial foam cell subtypes of the atherosclerotic plaque. Atherosclerosis was induced in hypercholesterolemic autophagy reporter mice (GFP-LC3 mice receiving PCSK9-AAV and fed a Western diet). Autophagic flux in aortic digests was assessed by quantifying GFP-LC3 fluorescence after ex vivo treatment with the autophagy inhibitor Bafilomycin A1 or vehicle. MΦ foam cells displayed functional autophagy as shown by an accumulation of GFP-LC3 upon autophagy inhibition. In contrast, VSMC foam cells did not similarly accumulate GFP-LC3 upon bafilomycin treatment, suggesting dysfunction autophagy in these cells. Additionally, immunostaining of late-stage aortic roots showed MΦ, but not VSMC, foam cells induction of the active autophagy marker pATG16L1. Cell culture studies of lipid loaded MΦ and VSMC corroborated this inability for VSMCs to initiate autophagy in vivo . In vitro , MΦ foam cells effluxed cholesterol to ApoA-I (14%) and HDL (50%), whereas VSMC foam cells minimally effluxed cholesterol to HDL (7%) but not apoA-I. However, unlike MΦ foam cells, VSMC efflux was pharmacologically induced by treatment with metformin. Our data therefore demonstrates a lack of functional autophagy in VSMC, as compared to MΦ foam cells, which impairs their ability to perform cholesterol efflux. This autophagy defect in VSMC foam cells can be increased by autophagy activation using metformin, highlighting both the importance of understanding cholesterol metabolism in all foam cell populations and a new avenue to treat atherosclerosis.

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 from different segments clustered not by anatomical proximity but by embryonic origin. 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.

Background and Aim: A central role of vascular smooth muscle cells (SMCs) in atherosclerosis has recently evolved that suggests causal genetic links to disease processes. Single cell sequencing studies of atherosclerotic plaques have identified multiple mesenchymal transition cell populations within the plaques. Here, we correlated cell fractions from plaques to coronary artery disease (CAD) related gene polymorphisms to identify novel SMC targets and study their influence on SMC function in atherosclerosis. Methods: Deconvolution analysis was performed on bulk microarray data from carotid plaques in the Biobank of Karolinska Endarterectomies (BiKE, n=127) using single cell sequencing data from coronary plaques (n=5). CAD-associated GWAS loci associated with mesenchymal cell fractions were identified, followed by functional analyses of these genes in SMC in vitro using migration, proliferation and apoptosis assays. Results: We identified 5 mesenchymal cell-specific genetic variants associated with CAD, BiKE patient symptomatology and gene expression eQTLs in BiKE plaque tissue and GTex normal arteries. These variants were harbored in genetic loci of ARNTL, LDLR, MIA3, PAK1 and ARHGAP15 . Microarray analysis revealed increased expression of ARHGAP15 and PAK1 and decreased levels of LDLR in carotid plaques compared with normal arteries (n=127 vs. 10 respectively, student’s t-test). Immunohistochemistry demonstrated increased expression of corresponding proteins in the fibrous cap of plaques compared to normal arteries (n=5). To investigate their function in SMCs, the genes were silenced using siRNAs followed by migration, proliferation and apoptosis assays. Preliminary results indicated that silencing of MIA3, LDLR and ARNTL inhibited SMC proliferation. Conclusions: The results of this project may reveal novel SMC-specific genetic links to the disease, which may serve as therapeutic targets to be explored for improved treatment of atherosclerosis.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC , however the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a novel SMC phenotype that governs both vascular remodeling and recruitment of inflammatory cells, as well as a chondromyocyte fate. The remodeling population was marked by uniquely high Mmp3 and Cxcl12 expression, and its appearance correlated with higher risk plaque features such as increased positive remodeling and macrophage content. Further, investigation of transcriptional mechanisms by which Smad3 alters SMC cell fate revealed novel roles for Hox and Sox transcription factors whose direct interaction with Smad3 regulate an extensive transcriptional program balancing remodeling and vascular extracellular matrix. These findings have significant implications for atherosclerotic and Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.

Atherosclerosis is characterized by a build-up of foam cells in the arterial wall, resulting from excess cholesterol uptake and accumulation of cytosolic lipid droplets (LDs). Autophagy has been shown to be atheroprotective in part by promoting the catabolism of LDs which liberates free cholesterol for efflux out of foam cells to cholesterol acceptors (ApoA-I or HDL) for removal from the body. Apart from macrophages (MΦ), vascular smooth muscle cells (VSMCs) comprise 50-70% of foam cells in the plaques. Unlike MΦ, the capacity of VSMC foam cells to metabolize cholesterol via autophagy is unknown. Here, w e performed a comparative analysis of the autophagic capacity and cholesterol efflux of arterial foam cell subtypes of the atherosclerotic plaque . Atherosclerosis was induced in hypercholesterolemic autophagy reporter mice (GFP-LC3 mice receiving PCSK9-AAV and fed a Western diet). Autophagic flux in aortic digests was assessed by quantifying GFP-LC3 fluorescence after ex vivo treatment with the autophagy inhibitor Bafilomycin A1 or vehicle. MΦ foam cells displayed functional autophagy as shown by an accumulation of GFP-LC3 upon autophagy inhibition. In contrast, VSMC foam cells did not similarly accumulate GFP-LC3 upon bafilomycin treatment, suggesting dysfunction autophagy in these cells. Additionally, immunostaining of late-stage aortic roots showed MΦ, but not VSMC, foam cells induction of the active autophagy marker pATG16L1. Cell culture studies of lipid loaded MΦ and VSMC corroborated this inability for VSMCs to initiate autophagy in vivo . In vitro , MΦ foam cells effluxed cholesterol to ApoA-I (14%) and HDL (50%), whereas VSMC foam cells minimally effluxed cholesterol to HDL (7%) but not apoA-I. However, unlike MΦ foam cells, VSMC efflux was pharmacologically induced by treatment with metformin. Our data therefore demonstrates a lack of functional autophagy in VSMC, as compared to MΦ foam cells, which impairs their ability to perform cholesterol efflux. This autophagy defect in VSMC foam cells can be increased by autophagy activation using metformin, highlighting both the importance of understanding cholesterol metabolism in all foam cell populations and a new avenue to treat atherosclerosis.

Cardiac pacemaker cells (CPCs) initiate the electric impulses that drive the rhythmic beating of the heart. CPCs reside in a heterogeneous, ECM-rich microenvironment termed the sinoatrial node (SAN). Surprisingly, little is known regarding the biochemical composition or mechanical properties of the SAN, and how the unique structural characteristics present in this region of the heart influence CPC function remains poorly understood. Here, we have identified that SAN development involves the construction of a “soft” macromolecular ECM that specifically encapsulates CPCs. In addition, we demonstrate that subjecting embryonic CPCs to substrate stiffnesses higher than those measured in vivo results in loss of coherent electrical oscillation and dysregulation of the HCN4 and NCX1 ion channels required for CPC automaticity. Collectively, these data indicate that local mechanics play a critical role in maintaining the embryonic CPC function while also quantitatively defining the range of material properties that are optimal for embryonic CPC maturation.

Smooth muscle cells (SMC) transition into a number of different phenotypes during atherosclerosis, including those that resemble fibroblasts and chondrocytes, and make up the majority of cells in atherosclerotic plaque. To better understand the epigenetic and transcriptional mechanisms that mediate these cell state changes, and how they relate to risk for coronary artery disease (CAD), we have investigated the causality and function of transcription factors (TFs) at genome wide associated loci. Employing in vitro CRISPR-Cas 9 genome and epigenome editing we show that complex genetic signals within a gene desert at 2q22.3 lie within novel smooth muscle long-distance enhancers for ZEB2 , a TF extensively studied in the context of epithelial mesenchymal transition (EMT) in development and cancer. Single-cell epigenetic and transcriptomic profiling demonstrated that Zeb2 regulates SMC phenotypic transition through chromatin remodeling that obviates accessibility and disrupts both Notch and TGFβ signaling, altering the epigenetic trajectory of SMC transitions. These changes resulted in an inability for transition SMC to turn off contractile programing and take on a fibroblast-like phenotype, and accelerated chondromyocyte differentiation, mirroring features of high-risk atherosclerotic plaques in human coronary arteries. Parallel epigenetic alterations are observed in human coronary artery smooth muscle cells, with ZEB binding motifs preferentially enriched for human genetic signals for coronary artery disease, affirming its role as a master epigenetic regulator of atherosclerosis pathology.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC biology can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC, but the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, more positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC transition cell state toward two fates: a novel SMC phenotype that governs both vascular remodeling and recruitment of inflammatory cells, as well as a chondromyocyte fate. The remodeling population was marked by uniquely high Mmp3 and Cxcl12 expression, and its appearance correlated with higher risk plaque features such as increased positive remodeling and macrophage content. Further, investigation of transcriptional mechanisms by which Smad3 alters SMC cell fate revealed novel roles for Hox and Sox transcription factors whose direct interaction with Smad3 regulate an extensive transcriptional program balancing remodeling and vascular extracellular matrix with significant implications for atherosclerotic and Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.