Single-Cell Gene-Regulatory Networks of Advanced Symptomatic Atherosclerosis.

BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.

Secreted Protein Profiling of Human Aortic Smooth Muscle Cells Identifies Vascular Disease Associations.

BACKGROUND: Smooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular disease, the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a contractile to a synthetic phenotype characterized by an increased proliferation, migration, production of ECM (extracellular matrix) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of cardiovascular disease, including coronary artery disease, stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies. METHODS: Using human aortic SMCs from 123 multiancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted liquid chromatography-tandem mass spectrometry-based proteomic analysis of the conditioned media. RESULTS: We measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 (latent-transforming growth factor beta-binding protein 1) in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions. CONCLUSIONS: Our results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.

Atherosclerotic Plaque Epigenetic Age Acceleration Predicts a Poor Prognosis and Is Associated With Endothelial-to-Mesenchymal Transition in Humans.

BACKGROUND: Epigenetic age estimators (clocks) are predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques is predictive for the risk of cardiovascular events. METHODS: Whole-genome DNA methylation of human carotid atherosclerotic plaques (n=485) and of blood (n=93) from the Athero-Express endarterectomy cohort was used to calculate epigenetic age acceleration (EAA). EAA was linked to clinical characteristics, plaque histology, and future cardiovascular events (n=136). We studied whole-genome DNA methylation and bulk and single-cell transcriptomics to uncover molecular mechanisms of plaque EAA. We experimentally confirmed our in silico findings using in vitro experiments in primary human coronary endothelial cells. RESULTS: Male and female patients with severe atherosclerosis had a median chronological age of 69 years. The median epigenetic age was 65 years in females (median EAA, -2.2 [interquartile range, -4.3 to 2.2] years) and 68 years in males (median EAA, -0.3 [interquartile range, -2.9 to 3.8] years). Patients with diabetes and a high body mass index had higher plaque EAA. Increased EAA of plaque predicted future events in a 3-year follow-up in a Cox regression model (univariate hazard ratio, 1.7; P=0.0034) and adjusted multivariate model (hazard ratio, 1.56; P=0.02). Plaque EAA predicted outcome independent of blood EAA (hazard ratio, 1.3; P=0.018) and of plaque hemorrhage (hazard ratio, 1.7; P=0.02). Single-cell RNA sequencing in plaque samples from 46 patients in the same cohort revealed smooth muscle and endothelial cells as important cell types in plaque EAA. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally confirmed by TGFß-triggered endothelial-to-mesenchymal transition inducing rapid epigenetic aging in coronary endothelial cells. CONCLUSIONS: Plaque EAA is a strong and independent marker of poor outcome in patients with severe atherosclerosis. Plaque EAA was linked to mesenchymal endothelial and smooth muscle cells. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally validated. Epigenetic aging mechanisms may provide new targets for treatments that reduce atherosclerosis complications.

Systems genetics analysis of human body fat distribution genes identifies adipocyte processes.

Excess abdominal fat is a sexually dimorphic risk factor for cardio-metabolic disease and is approximated by the waist-to-hip ratio adjusted for body mass index (WHRadjBMI). Whereas this trait is highly heritable, few causal genes are known. We aimed to identify novel drivers of WHRadjBMI using systems genetics. We used two independent cohorts of adipose tissue gene expression and constructed sex- and depot-specific Bayesian networks to model gene-gene interactions from 8,492 genes. Using key driver analysis, we identified genes that, in silico and putatively in vitro, regulate many others. 51-119 key drivers in each network were replicated in both cohorts. In other cell types, 23 of these genes are found in crucial adipocyte pathways: Wnt signaling or mitochondrial function. We overexpressed or down-regulated seven key driver genes in human subcutaneous pre-adipocytes. Key driver genes ANAPC2 and RSPO1 inhibited adipogenesis, whereas PSME3 increased adipogenesis. RSPO1 increased Wnt signaling activity. In differentiated adipocytes, MIGA1 and UBR1 down-regulation led to mitochondrial dysfunction. These five genes regulate adipocyte function, and we hypothesize that they regulate fat distribution.

Tobacco smoking is associated with sex- and plaque-type specific upregulation of CRLF1 in atherosclerotic lesions.

BACKGROUND AND AIMS: Tobacco smoking is a known risk factor for atherosclerotic disease, with more elevated risks in women compared to men. We hypothesized that atherosclerotic plaques from smokers show different gene expression patterns compared to non-smokers, in a sex-specific manner. METHODS: Gene expression data of 625 carotid plaques (151 females and 474 males) were analyzed for differential gene expression between current smokers (n = 226) and non-smokers (n = 399). All analyses were stratified by sex and by molecular plaque characteristics. Finally, we projected the activity of gene regulatory networks and utilized single-cell transcriptomics from 38 plaques (26 males and 12 females) to interpret the sex- and plaque-type specific signals. RESULTS: We observed higher expression levels of CRLF1 gene in atherosclerotic plaques from smokers compared to non-smokers (log2FC = 0.48, FDR = 0.012). CRLF1 upregulation was interacting with sex (p = 0.01) and was more pronounced in females (log2FC = 0.93, p = 1.53E-05) compared to males (log2FC = 0.35, p = 0.0018). Through single-cell RNA-seq analysis, we identified the highest CRLF1 expression within the transitioning and synthetic smooth muscle cell populations. CRLF1 expression was increased in fibro-inflammatory and fibro-cellular plaque types. Gene annotations pointed to increased expression of CRLF1 in networks with extracellular matrix related genes. CONCLUSIONS: Atherosclerotic plaques from current smokers show sex-dependent upregulation of smooth muscle cell gene CRLF1. This may explain the different contributions of smoking to cardiovascular risk in females.