Genetic Variation Determines PPARγ Function and Anti-diabetic Drug Response In Vivo.

SNPs affecting disease risk often reside in non-coding genomic regions. Here, we show that SNPs are highly enriched at mouse strain-selective adipose tissue binding sites for PPARγ, a nuclear receptor for anti-diabetic drugs. Many such SNPs alter binding motifs for PPARγ or cooperating factors and functionally regulate nearby genes whose expression is strain selective and imbalanced in heterozygous F1 mice. Moreover, genetically determined binding of PPARγ accounts for mouse strain-specific transcriptional effects of TZD drugs, providing proof of concept for personalized medicine related to nuclear receptor genomic occupancy. In human fat, motif-altering SNPs cause differential PPARγ binding, provide a molecular mechanism for some expression quantitative trait loci, and are risk factors for dysmetabolic traits in genome-wide association studies. One PPARγ motif-altering SNP is associated with HDL levels and other metabolic syndrome parameters. Thus, natural genetic variation in PPARγ genomic occupancy determines individual disease risk and drug response.

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.

Genetic Regulation of SMC Gene Expression and Splicing Predict Causal CAD Genes.

BACKGROUND: Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies have identified over 175 loci associated with CAD. The majority of these loci are in noncoding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we aimed to identify the subset of the CAD loci associated with the regulation of transcription in distinct SMC phenotypes. METHODS: We measured gene expression in SMCs isolated from the ascending aortas of 151 heart transplant donors of various genetic ancestries in quiescent or proliferative conditions and calculated the association of their expression and splicing with ~6.3 million imputed single-nucleotide polymorphism markers across the genome. RESULTS: We identified 4910 expression and 4412 splicing quantitative trait loci (sQTLs) representing regions of the genome associated with transcript abundance and splicing. A total of 3660 expression quantitative trait loci (eQTLs) had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 eQTLs were SMC-specific and sex-biased, respectively. We made these results available for public query on a user-friendly website. To identify the effector transcript(s) regulated by CAD loci, we used 4 distinct colocalization approaches. We identified 84 eQTL and 164 sQTL that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. One CAD locus colocalized with a sex-specific eQTL (TERF2IP), and another locus colocalized with SMC-specific eQTL (ALKBH8). The most significantly associated CAD locus, 9p21, was an sQTL for the long noncoding RNA CDKN2B-AS1, also known as ANRIL, in proliferative SMCs. CONCLUSIONS: Collectively, our results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.

FHL5 Controls Vascular Disease-Associated Gene Programs in Smooth Muscle Cells.

BACKGROUND: Genome-wide association studies have identified hundreds of loci associated with common vascular diseases, such as coronary artery disease, myocardial infarction, and hypertension. However, the lack of mechanistic insights for many GWAS loci limits their translation into the clinic. Among these loci with unknown functions is UFL1-four-and-a-half LIM (LIN-11, Isl-1, MEC-3) domain 5 (FHL5; chr6q16.1), which reached genome-wide significance in a recent coronary artery disease/ myocardial infarction GWAS meta-analysis. UFL1-FHL5 is also associated with several vascular diseases, consistent with the widespread pleiotropy observed for GWAS loci. METHODS: We apply a multimodal approach leveraging statistical fine-mapping, epigenomic profiling, and ex vivo analysis of human coronary artery tissues to implicate FHL5 as the top candidate causal gene. We unravel the molecular mechanisms of the cross-phenotype genetic associations through in vitro functional analyses and epigenomic profiling experiments in coronary artery smooth muscle cells. RESULTS: We prioritized FHL5 as the top candidate causal gene at the UFL1-FHL5 locus through expression quantitative trait locus colocalization methods. FHL5 gene expression was enriched in the smooth muscle cells and pericyte population in human artery tissues with coexpression network analyses supporting a functional role in regulating smooth muscle cell contraction. Unexpectedly, under procalcifying conditions, FHL5 overexpression promoted vascular calcification and dysregulated processes related to extracellular matrix organization and calcium handling. Lastly, by mapping FHL5 binding sites and inferring FHL5 target gene function using artery tissue gene regulatory network analyses, we highlight regulatory interactions between FHL5 and downstream coronary artery disease/myocardial infarction loci, such as FOXL1 and FN1 that have roles in vascular remodeling. CONCLUSIONS: Taken together, these studies provide mechanistic insights into the pleiotropic genetic associations of UFL1-FHL5. We show that FHL5 mediates vascular disease risk through transcriptional regulation of downstream vascular remodeling gene programs. These transacting mechanisms may explain a portion of the heritable risk for complex vascular diseases.

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.

Female Gene Networks Are Expressed in Myofibroblast-Like Smooth Muscle Cells in Vulnerable Atherosclerotic Plaques.

BACKGROUND: Women presenting with coronary artery disease more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. METHODS: Gene regulatory networks were created using RNAseq gene expression data from human carotid atherosclerotic plaques. The networks were prioritized based on sex bias, relevance for smooth muscle biology, and coronary artery disease genetic enrichment. Network expression was linked to histologically determined plaque phenotypes. In addition, their expression in plaque cell types was studied at single-cell resolution using single-cell RNAseq. Finally, their relevance for disease progression was studied in female and male Apoe-/- mice fed a Western diet for 18 and 30 weeks. RESULTS: Here, we identify multiple sex-stratified gene regulatory networks from human carotid atherosclerotic plaques. Prioritization of the female networks identified 2 main SMC gene regulatory networks in late-stage atherosclerosis. Single-cell RNA sequencing mapped these female networks to 2 SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like network was mostly expressed in plaques that were vulnerable in women. Finally, the mice ortholog of key driver gene MFGE8 (milk fat globule EGF and factor V/VIII domain containing) showed retained expression in advanced plaques from female mice but was downregulated in male mice during atherosclerosis progression. CONCLUSIONS: Female atherosclerosis is characterized by gene regulatory networks that are active in fibrous vulnerable plaques rich in myofibroblast-like SMCs.

Genes in human obesity loci are causal obesity genes in C. elegans.

Obesity and its associated metabolic syndrome are a leading cause of morbidity and mortality. Given the disease's heavy burden on patients and the healthcare system, there has been increased interest in identifying pharmacological targets for the treatment and prevention of obesity. Towards this end, genome-wide association studies (GWAS) have identified hundreds of human genetic variants associated with obesity. The next challenge is to experimentally define which of these variants are causally linked to obesity, and could therefore become targets for the treatment or prevention of obesity. Here we employ high-throughput in vivo RNAi screening to test for causality 293 C. elegans orthologs of human obesity-candidate genes reported in GWAS. We RNAi screened these 293 genes in C. elegans subject to two different feeding regimens: (1) regular diet, and (2) high-fructose diet, which we developed and present here as an invertebrate model of diet-induced obesity (DIO). We report 14 genes that promote obesity and 3 genes that prevent DIO when silenced in C. elegans. Further, we show that knock-down of the 3 DIO genes not only prevents excessive fat accumulation in primary and ectopic fat depots but also improves the health and extends the lifespan of C. elegans overconsuming fructose. Importantly, the direction of the association between expression variants in these loci and obesity in mice and humans matches the phenotypic outcome of the loss-of-function of the C. elegans ortholog genes, supporting the notion that some of these genes would be causally linked to obesity across phylogeny. Therefore, in addition to defining causality for several genes so far merely correlated with obesity, this study demonstrates the value of model systems compatible with in vivo high-throughput genetic screening to causally link GWAS gene candidates to human diseases.

A Platelet Function Modulator of Thrombin Activation Is Causally Linked to Cardiovascular Disease and Affects PAR4 Receptor Signaling.

Dual antiplatelet therapy reduces ischemic events in cardiovascular disease, but it increases bleeding risk. Thrombin receptors PAR1 and PAR4 are drug targets, but the role of thrombin in platelet aggregation remains largely unexplored in large populations. We performed a genome-wide association study (GWAS) of platelet aggregation in response to full-length thrombin, followed by clinical association analyses, Mendelian randomization, and functional characterization including iPSC-derived megakaryocyte and platelet experiments. We identified a single sentinel variant in the GRK5 locus (rs10886430-G, p = 3.0 × 10-42) associated with increased thrombin-induced platelet aggregation (ß = 0.70, SE = 0.05). We show that disruption of platelet GRK5 expression by rs10886430-G is associated with enhanced platelet reactivity. The proposed mechanism of a GATA1-driven megakaryocyte enhancer is confirmed in allele-specific experiments. Utilizing further data, we demonstrate that the allelic effect is highly platelet- and thrombin-specific and not likely due to effects on thrombin levels. The variant is associated with increased risk of cardiovascular disease outcomes in UK BioBank, most strongly with pulmonary embolism. The variant associates with increased risk of stroke in the MEGASTROKE, UK BioBank, and FinnGen studies. Mendelian randomization analyses in independent samples support a causal role for rs10886430-G in increasing risk for stroke, pulmonary embolism, and venous thromboembolism through its effect on thrombin-induced platelet reactivity. We demonstrate that G protein-coupled receptor kinase 5 (GRK5) promotes platelet activation specifically via PAR4 receptor signaling. GRK5 inhibitors in development for the treatment of heart failure and cancer could have platelet off-target deleterious effects. Common variants in GRK5 may modify clinical outcomes with PAR4 inhibitors, and upregulation of GRK5 activity or signaling in platelets may have therapeutic benefits.

Sex differences in human adipose tissue gene expression and genetic regulation involve adipogenesis.

Sex differences in adipose tissue distribution and function are associated with sex differences in cardiometabolic disease. While many studies have revealed sex differences in adipocyte cell signaling and physiology, there is a relative dearth of information regarding sex differences in transcript abundance and regulation. We investigated sex differences in subcutaneous adipose tissue transcriptional regulation using omic-scale data from ∼3000 geographically and ethnically diverse human samples. We identified 162 genes with robust sex differences in expression. Differentially expressed genes were implicated in oxidative phosphorylation and adipogenesis. We further determined that sex differences in gene expression levels could be related to sex differences in the genetics of gene expression regulation. Our analyses revealed sex-specific genetic associations, and this finding was replicated in a study of 98 inbred mouse strains. The genes under genetic regulation in human and mouse were enriched for oxidative phosphorylation and adipogenesis. Enrichment analysis showed that the associated genetic loci resided within binding motifs for adipogenic transcription factors (e.g., PPARG and EGR1). We demonstrated that sex differences in gene expression could be influenced by sex differences in genetic regulation for six genes (e.g., FADS1 and MAP1B). These genes exhibited dynamic expression patterns during adipogenesis and robust expression in mature human adipocytes. Our results support a role for adipogenesis-related genes in subcutaneous adipose tissue sex differences in the genetic and environmental regulation of gene expression.

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

[Figure: see text].

Sex-Stratified Gene Regulatory Networks Reveal Female Key Driver Genes of Atherosclerosis Involved in Smooth Muscle Cell Phenotype Switching.

BACKGROUND: Although sex differences in coronary artery disease are widely accepted with women developing more stable atherosclerosis than men, the underlying pathobiology of such differences remains largely unknown. In coronary artery disease, recent integrative systems biological studies have inferred gene regulatory networks (GRNs). Within these GRNs, key driver genes have shown great promise but have thus far been unidentified in women. METHODS: We generated sex-specific GRNs of the atherosclerotic arterial wall in 160 women and age-matched men in the STARNET study (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task). We integrated the female GRNs with single-cell RNA-sequencing data of the human atherosclerotic plaque and single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic smooth muscle cell (SMC) lineage-tracing mice. RESULTS: By comparing sex-specific GRNs, we observed clear sex differences in network activity within the atherosclerotic tissues. Genes more active in women were associated with mesenchymal cells and endothelial cells, whereas genes more active in men were associated with the immune system. We determined that key drivers of GRNs active in female coronary artery disease were predominantly found in (SMCs by single-cell sequencing of the human atherosclerotic plaques, and higher expressed in female plaque SMCs, as well. To study the functions of these female SMC key drivers in atherosclerosis, we examined single-cell RNA sequencing of advanced atherosclerotic lesions from wild type and Klf4 knockout atherosclerotic SMC lineage-tracing mice. The female key drivers were found to be expressed by phenotypically modulated SMCs and affected by Klf4, suggesting that sex differences in atherosclerosis involve phenotypic switching of plaque SMCs. CONCLUSIONS: Our systems approach provides novel insights into molecular mechanisms that underlie sex differences in atherosclerosis. To discover sex-specific therapeutic targets for atherosclerosis, an increased emphasis on sex-stratified approaches in the analysis of multi-omics data sets is warranted.

Adipose Tissue Gene Expression Associations Reveal Hundreds of Candidate Genes for Cardiometabolic Traits.

Genome-wide association studies (GWASs) have identified thousands of genetic loci associated with cardiometabolic traits including type 2 diabetes (T2D), lipid levels, body fat distribution, and adiposity, although most causal genes remain unknown. We used subcutaneous adipose tissue RNA-seq data from 434 Finnish men from the METSIM study to identify 9,687 primary and 2,785 secondary cis-expression quantitative trait loci (eQTL; <1 Mb from TSS, FDR < 1%). Compared to primary eQTL signals, secondary eQTL signals were located further from transcription start sites, had smaller effect sizes, and were less enriched in adipose tissue regulatory elements compared to primary signals. Among 2,843 cardiometabolic GWAS signals, 262 colocalized by LD and conditional analysis with 318 transcripts as primary and conditionally distinct secondary cis-eQTLs, including some across ancestries. Of cardiometabolic traits examined for adipose tissue eQTL colocalizations, waist-hip ratio (WHR) and circulating lipid traits had the highest percentage of colocalized eQTLs (15% and 14%, respectively). Among alleles associated with increased cardiometabolic GWAS risk, approximately half (53%) were associated with decreased gene expression level. Mediation analyses of colocalized genes and cardiometabolic traits within the 434 individuals provided further evidence that gene expression influences variant-trait associations. These results identify hundreds of candidate genes that may act in adipose tissue to influence cardiometabolic traits.

Genetic Regulation of Atherosclerosis-Relevant Phenotypes in Human Vascular Smooth Muscle Cells.

RATIONALE: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. OBJECTIVE: To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs. METHODS AND RESULTS: We quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3. The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans. CONCLUSIONS: Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

Pcpe2, a Novel Extracellular Matrix Protein, Regulates Adipocyte SR-BI-Mediated High-Density Lipoprotein Uptake.

Objective: To investigate the role of adipocyte Pcpe2 (procollagen C-endopeptidase enhancer 2) in SR-BI (scavenger receptor class BI)-mediated HDL-C (high-density lipoprotein cholesterol) uptake and contributions to adipose lipid storage. Approach and Results: Pcpe2, a glycoprotein devoid of intrinsic proteolytic activity, is believed to participate in extracellular protein-protein interactions, supporting SR-BI- mediated HDL-C uptake. In published studies, Pcpe2 deficiency increased the development of atherosclerosis by reducing SR-BI-mediated HDL-C catabolism, but the biological impact of this deficiency on adipocyte SR-BI-mediated HDL-C uptake is unknown. Differentiated cells from Ldlr-/-/Pcpe2-/- (Pcpe2-/-) mouse adipose tissue showed elevated SR-BI protein levels, but significantly reduced HDL-C uptake compared to Ldlr-/- (control) adipose tissue. SR-BI-mediated HDL-C uptake was restored by preincubation of cells with exogenous Pcpe2. In diet-fed mice lacking Pcpe2, significant reductions in visceral, subcutaneous, and brown adipose tissue mass were observed, despite elevations in plasma triglyceride and cholesterol concentrations. Significant positive correlations exist between adipose mass and Pcpe2 expression in both mice and humans. Conclusions: Overall, these findings reveal a novel and unexpected function for Pcpe2 in modulating SR-BI expression and function as it relates to adipose tissue expansion and cholesterol balance in both mice and humans.

CD47-blocking antibodies restore phagocytosis and prevent atherosclerosis.

Atherosclerosis is the disease process that underlies heart attack and stroke. Advanced lesions at risk of rupture are characterized by the pathological accumulation of diseased vascular cells and apoptotic cellular debris. Why these cells are not cleared remains unknown. Here we show that atherogenesis is associated with upregulation of CD47, a key anti-phagocytic molecule that is known to render malignant cells resistant to programmed cell removal, or 'efferocytosis'. We find that administration of CD47-blocking antibodies reverses this defect in efferocytosis, normalizes the clearance of diseased vascular tissue, and ameliorates atherosclerosis in multiple mouse models. Mechanistic studies implicate the pro-atherosclerotic factor TNF-α as a fundamental driver of impaired programmed cell removal, explaining why this process is compromised in vascular disease. Similar to recent observations in cancer, impaired efferocytosis appears to play a pathogenic role in cardiovascular disease, but is not a fixed defect and may represent a novel therapeutic target.

A proteomic atlas of the neointima identifies novel druggable targets for preventive therapy.

AIMS: In-stent restenosis is a complication after coronary stenting associated with morbidity and mortality. Here, we sought to investigate the molecular processes underlying neointima formation and to identify new treatment and prevention targets. METHODS AND RESULTS: Neointima formation was induced by wire injury in mouse femoral arteries. High-accuracy proteomic measurement of single femoral arteries to a depth of about 5000 proteins revealed massive proteome remodelling, with more than half of all proteins exhibiting expression differences between injured and non-injured vessels. We observed major changes in the composition of the extracellular matrix and cell migration processes. Among the latter, we identified the classical transient receptor potential channel 6 (TRPC6) to drive neointima formation. While Trpc6-/- mice presented reduced neointima formation compared to wild-type mice (1.44 ± 0.39 vs. 2.16 ± 0.48, P = 0.01), activating or repressing TRPC6 in human vascular smooth muscle cells resulted in increased [vehicle 156.9 ± 15.8 vs. 1-oleoyl-2-acetyl-sn-glycerol 179.1 ± 8.07 (103 pixels), P = 0.01] or decreased migratory capacity [vehicle 130.0 ± 26.1 vs. SAR7334 111.4 ± 38.0 (103 pixels), P = 0.04], respectively. In a cohort of individuals with angiographic follow-up (n = 3068, males: 69.9%, age: 59 ± 11 years, follow-up 217.1 ± 156.4 days), homozygous carriers of a common genetic variant associated with elevated TRPC6 expression were at increased risk of restenosis after coronary stenting (adjusted odds ratio 1.49, 95% confidence interval 1.08-2.05; P = 0.01). CONCLUSIONS: Our study provides a proteomic atlas of the healthy and injured arterial wall that can be used to define novel factors for therapeutic targeting. We present TRPC6 as an actionable target to prevent neointima formation secondary to vascular injury and stent implantation.

Assessing exposure effects on gene expression.

In observational genomics data sets, there is often confounding of the effect of an exposure on gene expression. To adjust for confounding when estimating the exposure effect, a common approach involves including potential confounders as covariates with the exposure in a regression model of gene expression. However, when the exposure and confounders interact to influence gene expression, the fitted regression model does not necessarily estimate the overall effect of the exposure. Using inverse probability weighting (IPW) or the parametric g-formula in these instances is straightforward to apply and yields consistent effect estimates. IPW can readily be integrated into a genomics data analysis pipeline with upstream data processing and normalization, while the g-formula can be implemented by making simple alterations to the regression model. The regression, IPW, and g-formula approaches to exposure effect estimation are compared herein using simulations; advantages and disadvantages of each approach are explored. The methods are applied to a case study estimating the effect of current smoking on gene expression in adipose tissue.

Colocalization of GWAS and eQTL signals at loci with multiple signals identifies additional candidate genes for body fat distribution.

Integration of genome-wide association study (GWAS) signals with expression quantitative trait loci (eQTL) studies enables identification of candidate genes. However, evaluating whether nearby signals may share causal variants, termed colocalization, is affected by the presence of allelic heterogeneity, different variants at the same locus impacting the same phenotype. We previously identified eQTL in subcutaneous adipose tissue from 770 participants in the Metabolic Syndrome in Men (METSIM) study and detected 15 eQTL signals that colocalized with GWAS signals for waist-hip ratio adjusted for body mass index (WHRadjBMI) from the Genetic Investigation of Anthropometric Traits consortium. Here, we reevaluated evidence of colocalization using two approaches, conditional analysis and the Bayesian test COLOC, and show that providing COLOC with approximate conditional summary statistics at multi-signal GWAS loci can reconcile disagreements in colocalization classification between the two tests. Next, we performed conditional analysis on the METSIM subcutaneous adipose tissue data to identify conditionally distinct or secondary eQTL signals. We used the two approaches to test for colocalization with WHRadjBMI GWAS signals and evaluated the differences in colocalization classification between the two tests. Through these analyses, we identified four GWAS signals colocalized with secondary eQTL signals for FAM13A, SSR3, GRB14 and FMO1. Thus, at loci with multiple eQTL and/or GWAS signals, analyzing each signal independently enabled additional candidate genes to be identified.

Defining data-driven primary transcript annotations with primaryTranscriptAnnotation in R.

SUMMARY: Nascent transcript measurements derived from run-on sequencing experiments are critical for the investigation of transcriptional mechanisms and regulatory networks. However, conventional mRNA gene annotations significantly differ from the boundaries of primary transcripts. New primary transcript annotations are needed to accurately interpret run-on data. We developed the primaryTranscriptAnnotation R package to infer the transcriptional start and termination sites of primary transcripts from genomic run-on data. We then used these inferred coordinates to annotate transcriptional units identified de novo. This package provides the novel utility to integrate data-driven primary transcript annotations with transcriptional unit coordinates identified in an unbiased manner. Highlighting the importance of using accurate primary transcript coordinates, we demonstrate that this new methodology increases the detection of differentially expressed transcripts and provides more accurate quantification of RNA polymerase pause indices. AVAILABILITY AND IMPLEMENTATION: https://github.com/WarrenDavidAnderson/genomicsRpackage/tree/master/primaryTranscriptAnnotation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.