Single-cell 'omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveal heterogeneity of endothelial subtype and response to activating perturbations.

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

BACKGROUND: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. METHODS: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq, chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. RESULTS: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial NO synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. CONCLUSIONS: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

Cytokine and Chemokine Receptor Profiles in Adipose Tissue Vasculature Unravel Endothelial Cell Responses in HIV.

Chronic systemic inflammation contributes to a substantially elevated risk of myocardial infarction in people living with HIV (PLWH). Endothelial cell dysfunction disrupts vascular homeostasis regulation, increasing the risk of vasoconstriction, inflammation, and thrombosis that contribute to cardiovascular disease. Our objective was to study the effects of plasma from PLWH on endothelial cell (EC) function, with the hypothesis that cytokines and chemokines are major drivers of EC activation. We first broadly phenotyped chemokine and cytokine receptor expression on arterial ECs, capillary ECs, venous ECs, and vascular smooth muscle cells (VSMCs) in adipose tissue in the subcutaneous adipose tissue of 59 PLWH using single cell transcriptomic analysis. We used CellChat to predict cell-cell interactions between ECs and other cells in the adipose tissue and Spearman correlation to measure the association between ECs and plasma cytokines. Finally, we cultured human arterial ECs (HAECs) in plasma-conditioned media from PLWH and performed bulk sequencing to study the direct effects ex-vivo. We observed that arterial and capillary ECs expressed higher interferon and tumor necrosis factor (TNF) receptors. Venous ECs had more interleukin (IL)-1R1 and ACKR1 receptors, and VSMCs had high significant IL-6R expression. CellChat predicted ligand-receptor interactions between adipose tissue immune cells as senders and capillary ECs as recipients in TNF-TNFRSF1A/B interactions. Chemokines expressed largely by capillary ECs were predicted to bind ACKR1 receptors on venous ECs. Beyond the adipose tissue, the proportion of venous ECs and VSMCs were positively plasma IL-6. In ex-vivo experiments, HAECs cultured with plasma-conditioned media from PLWH expressed transcripts that enriched for the TNF-α and reactive oxidative phosphorylation pathways. In conclusion, ECs demonstrate heterogeneity in cytokine and chemokine receptor expression. Further research is needed to fully elucidate the role of cytokines and chemokines in EC dysfunction and to develop effective therapeutic strategies.

Single cell 'omic profiles of human aortic endothelial cells in vitro and human atherosclerotic lesions ex vivo reveals heterogeneity of endothelial subtype and response to activating perturbations.

Objective: Endothelial cells (ECs), macrophages, and vascular smooth muscle cells (VSMCs) are major cell types in atherosclerosis progression, and heterogeneity in EC sub-phenotypes are becoming increasingly appreciated. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Approach and Results: To create an in vitro dataset to study human EC heterogeneity, multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs). To model pro-inflammatory and activating environments characteristic of the atherosclerotic microenvironment in vitro, HAECs from at least three donors were exposed to three distinct perturbations with their respective controls: transforming growth factor beta-2 (TGFB2), interleukin-1 beta (IL1B), and siRNA-mediated knock-down of the endothelial transcription factor ERG (siERG). To form a comprehensive in vivo/ex vivo dataset of human atherosclerotic cell types, meta-analysis of single cell transcriptomes across 17 human arterial specimens was performed. Two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and in vivo cell profiles. HAEC cultures were reproducibly populated by 4 major clusters with distinct pathway enrichment profiles: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Exposure to siERG, IL1B or TGFB2 elicited mostly distinct transcriptional and accessible chromatin responses. EC1 and EC2, the most canonically 'healthy' EC populations, were affected predominantly by siERG; the activated cluster EC3 was most responsive to IL1B; and the mesenchymal population EC4 was most affected by TGFB2. Quantitative comparisons between in vitro and in vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated SNPs from GWAS, suggesting these cell phenotypes harbor CAD-modulating mechanisms. Conclusion: Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here, which have been reported by others to be involved in the pathogenesis of atherosclerosis as well as induce endothelial-to-mesenchymal transition (EndMT), only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and in vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.

scDemultiplex: An iterative beta-binomial model-based method for accurate demultiplexing with hashtag oligos.

Single-cell sequencing have been widely used to characterize cellular heterogeneity. Sample multiplexing where multiple samples are pooled together for single-cell experiments, attracts wide attention due to its benefits of increasing capacity, reducing costs, and minimizing batch effects. To analyze multiplexed data, the first crucial step is to demultiplex, the process of assigning cells to individual samples. Inaccurate demultiplexing will create false cell types and result in misleading characterization. We propose scDemultiplex, which models hashtag oligo (HTO) counts with beta-binomial distribution and uses an iterative strategy for further refinement. Compared with seven existing demultiplexing approaches, scDemultiplex achieved great performance in both high-quality and low-quality data. Additionally, scDemultiplex can be combined with other approaches to improve their performance.

Functional noncoding SNPs in human endothelial cells fine-map vascular trait associations.

Functional consequences of genetic variation in the noncoding human genome are difficult to ascertain despite demonstrated associations to common, complex disease traits. To elucidate properties of functional noncoding SNPs with effects in human endothelial cells (ECs), we utilized our previous molecular quantitative trait locus (molQTL) analysis for transcription factor binding, chromatin accessibility, and H3K27 acetylation to nominate a set of likely functional noncoding SNPs. Together with information from genome-wide association studies (GWASs) for vascular disease traits, we tested the ability of 34,344 variants to perturb enhancer function in ECs using the highly multiplexed STARR-seq assay. Of these, 5711 variants validated, whose enriched attributes included: (1) mutations to TF binding motifs for ETS or AP-1 that are regulators of the EC state; (2) location in accessible and H3K27ac-marked EC chromatin; and (3) molQTL associations whereby alleles associate with differences in chromatin accessibility and TF binding across genetically diverse ECs. Next, using pro-inflammatory IL1B as an activator of cell state, we observed robust evidence (>50%) of context-specific SNP effects, underscoring the prevalence of noncoding gene-by-environment (GxE) effects. Lastly, using these cumulative data, we fine-mapped vascular disease loci and highlighted evidence suggesting mechanisms by which noncoding SNPs at two loci affect risk for pulse pressure/large artery stroke and abdominal aortic aneurysm through respective effects on transcriptional regulation of POU4F1 and LDAH Together, we highlight the attributes and context dependence of functional noncoding SNPs and provide new mechanisms underlying vascular disease risk.

Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs.

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq (chromatin immunoprecipitation sequencing). We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPß binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPß at these locations tolerated changes in spacing ranging from 5 bp increase to >30 bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.

Breast tumor stiffness instructs bone metastasis via maintenance of mechanical conditioning.

While the immediate and transitory response of breast cancer cells to pathological stiffness in their native microenvironment has been well explored, it remains unclear how stiffness-induced phenotypes are maintained over time after cancer cell dissemination in vivo. Here, we show that fibrotic-like matrix stiffness promotes distinct metastatic phenotypes in cancer cells, which are preserved after transition to softer microenvironments, such as bone marrow. Using differential gene expression analysis of stiffness-responsive breast cancer cells, we establish a multigenic score of mechanical conditioning (MeCo) and find that it is associated with bone metastasis in patients with breast cancer. The maintenance of mechanical conditioning is regulated by RUNX2, an osteogenic transcription factor, established driver of bone metastasis, and mitotic bookmarker that preserves chromatin accessibility at target gene loci. Using genetic and functional approaches, we demonstrate that mechanical conditioning maintenance can be simulated, repressed, or extended, with corresponding changes in bone metastatic potential.

Single-Cell Epigenomics and Functional Fine-Mapping of Atherosclerosis GWAS Loci.

[Figure: see text].

Genome-wide analysis identifies novel susceptibility loci for myocardial infarction.

AIMS: While most patients with myocardial infarction (MI) have underlying coronary atherosclerosis, not all patients with coronary artery disease (CAD) develop MI. We sought to address the hypothesis that some of the genetic factors which establish atherosclerosis may be distinct from those that predispose to vulnerable plaques and thrombus formation. METHODS AND RESULTS: We carried out a genome-wide association study for MI in the UK Biobank (n∼472 000), followed by a meta-analysis with summary statistics from the CARDIoGRAMplusC4D Consortium (n∼167 000). Multiple independent replication analyses and functional approaches were used to prioritize loci and evaluate positional candidate genes. Eight novel regions were identified for MI at the genome wide significance level, of which effect sizes at six loci were more robust for MI than for CAD without the presence of MI. Confirmatory evidence for association of a locus on chromosome 1p21.3 harbouring choline-like transporter 3 (SLC44A3) with MI in the context of CAD, but not with coronary atherosclerosis itself, was obtained in Biobank Japan (n∼165 000) and 16 independent angiography-based cohorts (n∼27 000). Follow-up analyses did not reveal association of the SLC44A3 locus with CAD risk factors, biomarkers of coagulation, other thrombotic diseases, or plasma levels of a broad array of metabolites, including choline, trimethylamine N-oxide, and betaine. However, aortic expression of SLC44A3 was increased in carriers of the MI risk allele at chromosome 1p21.3, increased in ischaemic (vs. non-diseased) coronary arteries, up-regulated in human aortic endothelial cells treated with interleukin-1ß (vs. vehicle), and associated with smooth muscle cell migration in vitro. CONCLUSIONS: A large-scale analysis comprising ∼831 000 subjects revealed novel genetic determinants of MI and implicated SLC44A3 in the pathophysiology of vulnerable plaques.

Systems Genetics in Human Endothelial Cells Identifies Non-coding Variants Modifying Enhancers, Expression, and Complex Disease Traits.

The identification of causal variants and mechanisms underlying complex disease traits in humans is important for the progress of human disease genetics; this requires finding strategies to detect functional regulatory variants in disease-relevant cell types. To achieve this, we collected genetic and transcriptomic data from the aortic endothelial cells of up to 157 donors and four epigenomic phenotypes in up to 44 human donors representing individuals of both sexes and three major ancestries. We found thousands of expression quantitative trait loci (eQTLs) at all ranges of effect sizes not detected by the Gene-Tissue Expression Project (GTEx) in human tissues, showing that novel biological relationships unique to endothelial cells (ECs) are enriched in this dataset. Epigenetic profiling enabled discovery of over 3,000 regulatory elements whose activity is modulated by genetic variants that most frequently mutated ETS, AP-1, and NF-kB binding motifs, implicating these motifs as governors of EC regulation. Using CRISPR interference (CRISPRi), allele-specific reporter assays, and chromatin conformation capture, we validated candidate enhancer variants located up to 750 kb from their target genes, VEGFC, FGD6, and KIF26B. Regulatory SNPs identified were enriched in coronary artery disease (CAD) loci, and this result has specific implications for PECAM-1, FES, and AXL. We also found significant roles for EC regulatory variants in modifying the traits pulse pressure, blood protein levels, and monocyte count. Lastly, we present two unlinked SNPs in the promoter of MFAP2 that exhibit pleiotropic effects on human disease traits. Together, this supports the possibility that genetic predisposition for complex disease is manifested through the endothelium.

Genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics.

Biomechanical cues dynamically control major cellular processes, but whether genetic variants actively participate in mechanosensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies (GWAS) have identified chromosome 1p32.2 as strongly associated with CAD/IS; however, the causal mechanism related to this locus remains unknown. Using statistical analyses, assay of transposase accessible chromatin with whole-genome sequencing (ATAC-seq), H3K27ac/H3K4me2 ChIP with whole-genome sequencing (ChIP-seq), and CRISPR interference in human aortic endothelial cells (HAECs), our results demonstrate that rs17114036, a common noncoding polymorphism at 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR-Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional shear stress by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus (caQTL) mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays demonstrate that CAD/IS-protective allele at rs17114036 in PLPP3 intron 5 confers increased endothelial enhancer activity. ChIP-PCR and luciferase assays show that CAD/IS-protective allele at rs17114036 creates a binding site for transcription factor Krüppel-like factor 2 (KLF2), which increases the enhancer activity under unidirectional flow. These results demonstrate that a human SNP contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cis-regulatory elements provide a previously unappreciated layer of regulatory control in cellular mechanosensing mechanisms.

Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells.

Endothelial cells (ECs) are critical determinants of vascular homeostasis and inflammation, but transcriptional mechanisms specifying their identities and functional states remain poorly understood. Here, we report a genome-wide assessment of regulatory landscapes of primary human aortic endothelial cells (HAECs) under basal and activated conditions, enabling inference of transcription factor networks that direct homeostatic and pro-inflammatory programs. We demonstrate that 43% of detected enhancers are EC-specific and contain SNPs associated to cardiovascular disease and hypertension. We provide evidence that AP1, ETS, and GATA transcription factors play key roles in HAEC transcription by co-binding enhancers associated with EC-specific genes. We further demonstrate that exposure of HAECs to oxidized phospholipids or pro-inflammatory cytokines results in signal-specific alterations in enhancer landscapes and associate with coordinated binding of CEBPD, IRF1, and NFκB. Collectively, these findings identify cis-regulatory elements and corresponding trans-acting factors that contribute to EC identity and their specific responses to pro-inflammatory stimuli.