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Summary: Genome-wide association studies (GWASs) have identified hundreds of risk loci for coronary artery disease (CAD). However, non-European populations are underrepresented in GWASs, and the causal gene-regulatory mechanisms of these risk loci during atherosclerosis remain unclear. We incorporated local ancestry and haplotypes to identify quantitative trait loci for expression (eQTLs) and splicing (sQTLs) in coronary arteries from 138 ancestrally diverse Americans. Of 2,132 eQTL-associated genes (eGenes), 47% were previously unreported in coronary artery; 19% exhibited cell-type-specific expression. Colocalization revealed subgroups of eGenes unique to CAD and blood pressure GWAS. Fine-mapping highlighted additional eGenes, including TBX20 and IL5. We also identified sQTLs for 1,690 genes, among which TOR1AIP1 and ULK3 sQTLs demonstrated the importance of evaluating splicing to accurately identify disease-relevant isoform expression. Our work provides a patient-derived coronary artery eQTL resource and exemplifies the need for diverse study populations and multifaceted approaches to characterize gene regulation in disease processes.

Epigenetically switched, proliferative vascular smooth muscle cells (SMCs) form neointima, engendering stenotic diseases. Histone-3 lysine-27 trimethylation (H3K27me3) and acetylation (H3K27ac) marks are associated with gene repression and activation, respectively. The polycomb protein embryonic ectoderm development (EED) reads H3K27me3 and also enhances its deposition, hence is a canonical gene repressor. However, herein we found an unexpected role for EED in activating the bona fide pro-proliferative gene Ccnd1 (cyclinD1). EED overexpression in SMCs increased Ccnd1 mRNA, seemingly contradicting its gene-repressing function. However, consistently, EED co-immunoprecipitated with gene-activating H3K27ac reader BRD4, and they co-occupied at both mitogen-activated Ccnd1 and mitogen-repressed P57 (bona fide anti-proliferative gene), as indicated by chromatin immunoprecipitation qPCR. These results were abolished by an inhibitor of either the EED/H3K27me3 or BRD4/H3K27ac reader function. In accordance, elevating BRD4 increased H3K27me3. In vivo, while EED was upregulated in rat and human neointimal lesions, selective EED inhibition abated angioplasty-induced neointima and reduced cyclinD1 in rat carotid arteries. Thus, results uncover a previously unknown role for EED in Ccnd1 activation, likely via its cooperativity with BRD4 that enhances each other’s reader function; i.e., activating pro-proliferative Ccnd1 while repressing anti-proliferative P57. As such, this study confers mechanistic implications for the epigenetic intervention of neointimal pathology.

Summary: Coronary artery disease (CAD) is characterized by atherosclerotic plaque formation in the arterial wall. CAD progression involves complex interactions and phenotypic plasticity among vascular and immune cell lineages. Single-cell RNA-seq (scRNA-seq) studies have highlighted lineage-specific transcriptomic signatures, but human cell phenotypes remain controversial. Here, we perform an integrated meta-analysis of 22 scRNA-seq libraries to generate a comprehensive map of human atherosclerosis with 118,578 cells. Besides characterizing granular cell-type diversity and communication, we leverage this atlas to provide insights into smooth muscle cell (SMC) modulation. We integrate genome-wide association study data and uncover a critical role for modulated SMC phenotypes in CAD, myocardial infarction, and coronary calcification. Finally, we identify fibromyocyte/fibrochondrogenic SMC markers (LTBP1 and CRTAC1) as proxies of atherosclerosis progression and validate these through omics and spatial imaging analyses. Altogether, we create a unified atlas of human atherosclerosis informing cell state-specific mechanistic and translational studies of cardiovascular diseases.

Vascular calcification (VC) is concomitant with atherosclerosis, yet it remains uncertain why rupture-prone high-risk plaques do not typically show extensive calcification. Intraplaque hemorrhage (IPH) deposits erythrocyte-derived cholesterol, enlarging the necrotic core and promoting high-risk plaque development. Pro-atherogenic CD163+ alternative macrophages engulf hemoglobin:haptoglobin (HH) complexes at IPH sites. However, their role in VC has never been examined to our knowledge. Here we show, in human arteries, the distribution of CD163+ macrophages correlated inversely with VC. In vitro experiments using vascular smooth muscle cells (VSMCs) cultured with HH-exposed human macrophage — M(Hb) — supernatant reduced calcification, while arteries from ApoE–/– CD163–/– mice showed greater VC. M(Hb) supernatant–exposed VSMCs showed activated NF-κB, while blocking NF-κB attenuated the anticalcific effect of M(Hb) on VSMCs. CD163+ macrophages altered VC through NF-κB–induced transcription of hyaluronan synthase (HAS), an enzyme that catalyzes the formation of the extracellular matrix glycosaminoglycan, hyaluronan, within VSMCs. M(Hb) supernatants enhanced HAS production in VSMCs, while knocking down HAS attenuated its anticalcific effect. NF-κB blockade in ApoE–/– mice reduced hyaluronan and increased VC. In human arteries, hyaluronan and HAS were increased in areas of CD163+ macrophage presence. Our findings highlight an important mechanism by which CD163+ macrophages inhibit VC through NF-κB–induced HAS augmentation and thus promote the high-risk plaque development.

Abstract Background Epigenome analysis relies on defined sets of genomic regions output by widely used assays such as ChIP-seq and ATAC-seq. Statistical analysis and visualization of genomic region sets is essential to answer biological questions in gene regulation. As the epigenomics community continues generating data, there will be an increasing need for software tools that can efficiently deal with more abundant and larger genomic region sets. Here, we introduce GenomicDistributions, an R package for fast and easy summarization and visualization of genomic region data. Results GenomicDistributions offers a broad selection of functions to calculate properties of genomic region sets, such as feature distances, genomic partition overlaps, and more. GenomicDistributions functions are meticulously optimized for best-in-class speed and generally outperform comparable functions in existing R packages. GenomicDistributions also offers plotting functions that produce editable ggplot objects. All GenomicDistributions functions follow a uniform naming scheme and can handle either single or multiple region set inputs. Conclusions GenomicDistributions offers a fast and scalable tool for exploratory genomic region set analysis and visualization. GenomicDistributions excels in user-friendliness, flexibility of outputs, breadth of functions, and computational performance. GenomicDistributions is available from Bioconductor ( https://bioconductor.org/packages/release/bioc/html/GenomicDistributions.html ).

Single-cell RNA-seq (scRNA-seq) is a powerful genomics technology to interrogate the cellular composition and behaviors of complex systems. While the number of scRNA-seq datasets and available computational analysis tools have grown exponentially, there are limited systematic data sharing strategies to allow rapid exploration and re-analysis of single-cell datasets, particularly in the cardiovascular field. We previously introduced PlaqView, an open-source web portal for the exploration and analysis of published atherosclerosis single-cell datasets. Now, we introduce PlaqView 2.0 (www.plaqview.com), which provides expanded features and functionalities as well as additional cardiovascular single-cell datasets. We showcase improved PlaqView functionality, backend data processing, user-interface, and capacity. PlaqView brings new or improved tools to explore scRNA-seq data, including gene query, metadata browser, cell identity prediction, ad hoc RNA-trajectory analysis, and drug-gene interaction prediction. PlaqView serves as one of the largest central repositories for cardiovascular single-cell datasets, which now includes data from human aortic aneurysm, gene-specific mouse knockouts, and healthy references. PlaqView 2.0 brings advanced tools and high-performance computing directly to users without the need for any programming knowledge. Lastly, we outline steps to generalize and repurpose PlaqView's framework for single-cell datasets from other fields.

Fat stored in the form of lipid droplets has long been considered a defining characteristic of cytoplasm. However, recent studies have shown that nuclear lipid droplets occur in multiple cells and tissues, including in human patients with fatty liver disease. The function(s) of stored fat in the nucleus has not been determined, and it is possible that nuclear fat is beneficial in some situations. Conversely, nuclear lipid droplets might instead be deleterious by disrupting nuclear organization or triggering aggregation of hydrophobic proteins. We show here that nuclear lipid droplets occur normally in C. elegans intestinal cells and germ cells, but appear to be associated with damage only in the intestine. Lipid droplets in intestinal nuclei can be associated with novel bundles of microfilaments (nuclear actin) and membrane tubules that might have roles in damage repair. To increase the normal, low frequency of nuclear lipid droplets in wild-type animals, we used a forward genetic screen to isolate mutants with abnormally large or abundant nuclear lipid droplets. Genetic analysis and cloning of three such mutants showed that the genes encode the lipid regulator SEIP-1/seipin, the inner nuclear membrane protein NEMP-1/Nemp1/TMEM194A, and a component of COPI vesicles called COPA-1/α-COP. We present several lines of evidence that the nuclear lipid droplet phenotype of copa-1 mutants results from a defect in retrieving mislocalized membrane proteins that normally reside in the endoplasmic reticulum. The seip-1 mutant causes most germ cells to have nuclear lipid droplets, the largest of which occupy more than a third of the nuclear volume. Nevertheless, the nuclear lipid droplets do not trigger apoptosis, and the germ cells differentiate into gametes that produce viable, healthy progeny. Thus, our results suggest that nuclear lipid droplets are detrimental to intestinal nuclei, but have no obvious deleterious effect on germ nuclei.