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.

An integrative multiomic network model links lipid metabolism to glucose regulation in coronary artery disease.

Elevated plasma cholesterol and type 2 diabetes (T2D) are associated with coronary artery disease (CAD). Individuals treated with cholesterol-lowering statins have increased T2D risk, while individuals with hypercholesterolemia have reduced T2D risk. We explore the relationship between lipid and glucose control by constructing network models from the STARNET study with sequencing data from seven cardiometabolic tissues obtained from CAD patients during coronary artery by-pass grafting surgery. By integrating gene expression, genotype, metabolomic, and clinical data, we identify a glucose and lipid determining (GLD) regulatory network showing inverse relationships with lipid and glucose traits. Master regulators of the GLD network also impact lipid and glucose levels in inverse directions. Experimental inhibition of one of the GLD network master regulators, lanosterol synthase (LSS), in mice confirms the inverse relationships to glucose and lipid levels as predicted by our model and provides mechanistic insights.

Unraveling tumor-immune heterogeneity in advanced ovarian cancer uncovers immunogenic effect of chemotherapy.

In metastatic cancer, the degree of heterogeneity of the tumor microenvironment (TME) and its molecular underpinnings remain largely unstudied. To characterize the tumor-immune interface at baseline and during neoadjuvant chemotherapy (NACT) in high-grade serous ovarian cancer (HGSOC), we performed immunogenomic analysis of treatment-naive and paired samples from before and after treatment with chemotherapy. In treatment-naive HGSOC, we found that immune-cell-excluded and inflammatory microenvironments coexist within the same individuals and within the same tumor sites, indicating ubiquitous variability in immune cell infiltration. Analysis of TME cell composition, DNA copy number, mutations and gene expression showed that immune cell exclusion was associated with amplification of Myc target genes and increased expression of canonical Wnt signaling in treatment-naive HGSOC. Following NACT, increased natural killer (NK) cell infiltration and oligoclonal expansion of T cells were detected. We demonstrate that the tumor-immune microenvironment of advanced HGSOC is intrinsically heterogeneous and that chemotherapy induces local immune activation, suggesting that chemotherapy can potentiate the immunogenicity of immune-excluded HGSOC tumors.

Clonally expanding smooth muscle cells promote atherosclerosis by escaping efferocytosis and activating the complement cascade.

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of "dedifferentiated" vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don't eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

Systems Pharmacology Identifies an Arterial Wall Regulatory Gene Network Mediating Coronary Artery Disease Side Effects of Antiretroviral Therapy.

BACKGROUND: Antiretroviral therapy (ART) for HIV infection increases risk for coronary artery disease (CAD), presumably by causing dyslipidemia and increased atherosclerosis. We applied systems pharmacology to identify and validate specific regulatory gene networks through which ART drugs may promote CAD. METHODS: Transcriptional responses of human cell lines to 15 ART drugs retrieved from the Library of Integrated Cellular Signatures (overall 1127 experiments) were used to establish consensus ART gene/transcriptional signatures. Next, enrichments of differentially expressed genes and gene-gene connectivity within these ART-consensus signatures were sought in 30 regulatory gene networks associated with CAD and CAD-related phenotypes in the Stockholm Atherosclerosis Gene Expression study. RESULTS: Ten of 15 ART signatures were significantly enriched both for differential expression and connectivity in a specific atherosclerotic arterial wall regulatory gene network (AR-RGN) causal for CAD involving RNA processing genes. An atherosclerosis in vitro model of cholestryl ester-loaded foam cells was then used for experimental validation. Treatments of these foam cells with ritonavir, nelfinavir, and saquinavir at least doubled cholestryl ester accumulation ( P=0.02, 0.0009, and 0.02, respectively), whereas RNA silencing of the AR-RGN top key driver, PQBP1 (polyglutamine binding protein 1), significantly curbed cholestryl ester accumulation following treatment with any of these ART drugs by >37% ( P<0.05). CONCLUSIONS: By applying a novel systems pharmacology data analysis framework, 3 commonly used ARTs (ritonavir, nelfinavir, and saquinavir) were found altering the activity of AR-RGN, a regulatory gene network promoting foam cell formation and risk of CAD. Targeting AR-RGN or its top key driver PQBP1 may help reduce CAD side effects of these ART drugs.

Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus.

Recent genome-wide association studies (GWAS) have identified multiple new loci which appear to alter coronary artery disease (CAD) risk via arterial wall-specific mechanisms. One of the annotated genes encodes LMOD1 (Leiomodin 1), a member of the actin filament nucleator family that is highly enriched in smooth muscle-containing tissues such as the artery wall. However, it is still unknown whether LMOD1 is the causal gene at this locus and also how the associated variants alter LMOD1 expression/function and CAD risk. Using epigenomic profiling we recently identified a non-coding regulatory variant, rs34091558, which is in tight linkage disequilibrium (LD) with the lead CAD GWAS variant, rs2820315. Herein we demonstrate through expression quantitative trait loci (eQTL) and statistical fine-mapping in GTEx, STARNET, and human coronary artery smooth muscle cell (HCASMC) datasets, rs34091558 is the top regulatory variant for LMOD1 in vascular tissues. Position weight matrix (PWM) analyses identify the protective allele rs34091558-TA to form a conserved Forkhead box O3 (FOXO3) binding motif, which is disrupted by the risk allele rs34091558-A. FOXO3 chromatin immunoprecipitation and reporter assays show reduced FOXO3 binding and LMOD1 transcriptional activity by the risk allele, consistent with effects of FOXO3 downregulation on LMOD1. LMOD1 knockdown results in increased proliferation and migration and decreased cell contraction in HCASMC, and immunostaining in atherosclerotic lesions in the SMC lineage tracing reporter mouse support a key role for LMOD1 in maintaining the differentiated SMC phenotype. These results provide compelling functional evidence that genetic variation is associated with dysregulated LMOD1 expression/function in SMCs, together contributing to the heritable risk for CAD.

Macrophage Trafficking, Inflammatory Resolution, and Genomics in Atherosclerosis: JACC Macrophage in CVD Series (Part 2).

Atherosclerosis is characterized by the retention of modified lipoproteins in the arterial wall. These modified lipoproteins activate resident macrophages and the recruitment of monocyte-derived cells, which differentiate into mononuclear phagocytes that ingest the deposited lipoproteins to become "foam cells": a hallmark of this disease. In this Part 2 of a 4-part review series covering the macrophage in cardiovascular disease, we critically review the contributions and relevant pathobiology of monocytes, macrophages, and foam cells as relevant to atherosclerosis. We also review evidence that via various pathways, a failure of the resolution of inflammation is an additional key aspect of this disease process. Finally, we consider the likely role played by genomics and biological networks in controlling the macrophage phenotype in atherosclerosis. Collectively, these data provide substantial insights on the atherosclerotic process, while concurrently offering numerous molecular and genomic candidates that appear to hold great promise for selective targeting as clinical therapies.

CD90 Identifies Adventitial Mesenchymal Progenitor Cells in Adult Human Medium- and Large-Sized Arteries.

Mesenchymal stem cells (MSCs) reportedly exist in a vascular niche occupying the outer adventitial layer. However, these cells have not been well characterized in vivo in medium- and large-sized arteries in humans, and their potential pathological role is unknown. To address this, healthy and diseased arterial tissues were obtained as surplus surgical specimens and freshly processed. We identified that CD90 marks a rare adventitial population that co-expresses MSC markers including PDGFRα, CD44, CD73, and CD105. However, unlike CD90, these additional markers were widely expressed by other cells. Human adventitial CD90+ cells fulfilled standard MSC criteria, including plastic adherence, spindle morphology, passage ability, colony formation, and differentiation into adipocytes, osteoblasts, and chondrocytes. Phenotypic and transcriptomic profiling, as well as adoptive transfer experiments, revealed a potential role in vascular disease pathogenesis, with the transcriptomic disease signature of these cells being represented in an aortic regulatory gene network that is operative in atherosclerosis.

JCAD, a Gene at the 10p11 Coronary Artery Disease Locus, Regulates Hippo Signaling in Endothelial Cells.

Objective- A large number of genetic loci have been associated with risk of coronary artery disease (CAD) through genome-wide association studies, however, for most loci the underlying biological mechanism is unknown. Determining the molecular pathways and cellular processes affected by these loci will provide new insights into CAD pathophysiology and may lead to new therapies. The CAD-associated variants at 10p11.23 fall in JCAD, which encodes an endothelial junction protein, however, its molecular function in endothelial cells is not known. In this study, we characterize the molecular role of JCAD (junctional cadherin 5 associated) in endothelial cells. Approach and Results- We show that JCAD knockdown in endothelial cells affects key phenotypes related to atherosclerosis including proliferation, migration, apoptosis, tube formation, and monocyte binding. We demonstrate that JCAD interacts with LATS2 (large tumor suppressor kinase 2) and negatively regulates Hippo signaling leading to increased activity of YAP (yes-associated protein), the transcriptional effector of the pathway. We also show by double siRNA knockdown that the phenotypes caused by JCAD knockdown require LATS2 and that JCAD is involved in transmission of RhoA-mediated signals into the Hippo pathway. In human tissues, we find that the CAD-associated lead variant, rs2487928, is associated with expression of JCAD in arteries, including atherosclerotic arteries. Gene co-expression analyses across disease-relevant tissues corroborate our phenotypic findings and support the link between JCAD and Hippo signaling. Conclusions- Our results show that JCAD negatively regulates Hippo signaling in endothelial cells and we suggest that JCAD contributes to atherosclerosis by mediating YAP activity and contributing to endothelial dysfunction.

Integration of pan-cancer transcriptomics with RPPA proteomics reveals mechanisms of epithelial-mesenchymal transition.

Integrating data from multiple regulatory layers across cancer types could elucidate additional mechanisms of oncogenesis. Using antibody-based protein profiling of 736 cancer cell lines, along with matching transcriptomic data, we show that pan-cancer bimodality in the amounts of mRNA, protein, and protein phosphorylation reveals mechanisms related to the epithelial-mesenchymal transition (EMT). Based on the bimodal expression of E-cadherin, we define an EMT signature consisting of 239 genes, many of which were not previously associated with EMT. By querying gene expression signatures collected from cancer cell lines after small-molecule perturbations, we identify enrichment for histone deacetylase (HDAC) inhibitors as inducers of EMT, and kinase inhibitors as mesenchymal-to-epithelial transition (MET) promoters. Causal modeling of protein-based signaling identifies putative drivers of EMT. In conclusion, integrative analysis of pan-cancer proteomic and transcriptomic data reveals key regulatory mechanisms of oncogenic transformation.

The Library of Integrated Network-Based Cellular Signatures NIH Program: System-Level Cataloging of Human Cells Response to Perturbations.

The Library of Integrated Network-Based Cellular Signatures (LINCS) is an NIH Common Fund program that catalogs how human cells globally respond to chemical, genetic, and disease perturbations. Resources generated by LINCS include experimental and computational methods, visualization tools, molecular and imaging data, and signatures. By assembling an integrated picture of the range of responses of human cells exposed to many perturbations, the LINCS program aims to better understand human disease and to advance the development of new therapies. Perturbations under study include drugs, genetic perturbations, tissue micro-environments, antibodies, and disease-causing mutations. Responses to perturbations are measured by transcript profiling, mass spectrometry, cell imaging, and biochemical methods, among other assays. The LINCS program focuses on cellular physiology shared among tissues and cell types relevant to an array of diseases, including cancer, heart disease, and neurodegenerative disorders. This Perspective describes LINCS technologies, datasets, tools, and approaches to data accessibility and reusability.

Dynamic Rearrangement of Cell States Detected by Systematic Screening of Sequential Anticancer Treatments.

Signaling networks are nonlinear and complex, involving a large ensemble of dynamic interaction states that fluctuate in space and time. However, therapeutic strategies, such as combination chemotherapy, rarely consider the timing of drug perturbations. If we are to advance drug discovery for complex diseases, it will be essential to develop methods capable of identifying dynamic cellular responses to clinically relevant perturbations. Here, we present a Bayesian dose-response framework and the screening of an oncological drug matrix, comprising 10,000 drug combinations in melanoma and pancreatic cancer cell lines, from which we predict sequentially effective drug combinations. Approximately 23% of the tested combinations showed high-confidence sequential effects (either synergistic or antagonistic), demonstrating that cellular perturbations of many drug combinations have temporal aspects, which are currently both underutilized and poorly understood.

MicroRNA-210, MicroRNA-331, and MicroRNA-7 Are Differentially Regulated in Treated HIV-1-Infected Individuals and Are Associated With Markers of Systemic Inflammation.

OBJECTIVE: Inflammation may contribute to an increased risk of cardiovascular disease (CVD) in HIV-1 infection. MicroRNAs (miRNAs) are involved in the regulation of inflammation. In treated HIV-1-infected individuals, we aimed to identify differentially expressed miRNAs with known roles in inflammation and CVD risk and to investigate associations between these and systemic inflammation. METHODS: In a screening cohort including 14 HIV-1-infected individuals and 9 uninfected controls, microarray profiling was performed using peripheral blood mononuclear cells (PBMCs). Differentially regulated miRNAs previously related to inflammation and CVD were validated using real-time quantitative reverse-transcription polymerase chain reaction in 26 HIV-1-infected individuals and 20 uninfected controls. Validated miRNAs were measured in PBMCs, CD4 and CD8 T cells. Interleukin-6, tumor necrosis factor-alpha, high-sensitivity C-reactive protein, lipopolysaccharide (LPS), cytomegalovirus immunoglobulin G, lipids, and fasting glucose were measured, and associations with validated miRNAs were assessed with multiple linear regression analysis. RESULTS: Upregulation of miR-210, miR-7, and miR-331 was found in PBMCs from HIV-1-infected individuals when compared with those from uninfected controls (P < 0.005). In contrast, miR-210 and miR-331 were downregulated in CD8 T cells. In multivariate analysis, miR-210 in CD8 T cells was negatively associated with LPS (P = 0.023) and triglycerides (P = 0.003) but positively associated with tumor necrosis factor-alpha (P = 0.004). MiR-7 in PBMC was positively associated with interleukin-6 (P = 0.025) and fasting glucose (P = 0.005), whereas miR-331 was negatively associated with LPS (P = 0.006). In PBMCs from HIV-1-infected individuals with low cytomegalovirus immunoglobulin G, miR-7, miR-29a, miR-221, and miR-222 were downregulated. CONCLUSION: In 2 independent cohorts, miR-210, miR-7, and miR-331 were differentially regulated in treated HIV-1-infected individuals and associated with markers of systemic inflammation.