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.

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.

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 nearly 19 000 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 (SYNTAX/Duke scores). 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 vascular 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-stage, 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.

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 diseases (CVD), 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 extracellular matrix (ECM) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of CVD, including coronary artery disease (CAD), 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 (GWAS). Methods: Using human aortic SMCs from 123 multi-ancestry 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 (LC-MS/MS)-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 (pQTL) 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 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.

Sex differences in the genetic and molecular mechanisms of coronary artery disease.

Sex differences in coronary artery disease (CAD) presentation, risk factors and prognosis have been widely studied. Similarly, studies on atherosclerosis have shown prominent sex differences in plaque biology. Our understanding of the underlying genetic and molecular mechanisms that drive these differences remains fragmented and largely understudied. Through reviewing genetic and epigenetic studies, we identified more than 40 sex-differential candidate genes (13 within known CAD loci) that may explain, at least in part, sex differences in vascular remodeling, lipid metabolism and endothelial dysfunction. Studies with transcriptomic and single-cell RNA sequencing data from atherosclerotic plaques highlight potential sex differences in smooth muscle cell and endothelial cell biology. Especially, phenotypic switching of smooth muscle cells seems to play a crucial role in female atherosclerosis. This matches the known sex differences in atherosclerotic phenotypes, with men being more prone to lipid-rich plaques, while women are more likely to develop fibrous plaques with endothelial dysfunction. To unravel the complex mechanisms that drive sex differences in CAD, increased statistical power and adjustments to study designs and analysis strategies are required. This entails increasing inclusion rates of women, performing well-defined sex-stratified analyses and the integration of multi-omics data.

Systems genetics analysis of human body fat distribution genes identifies Wnt signaling and mitochondrial activity in adipocytes.

BACKGROUND: Excess fat in the abdomen is a sexually dimorphic risk factor for cardio-metabolic disease. The relative storage between abdominal and lower-body subcutaneous adipose tissue depots is approximated by the waist-to-hip ratio adjusted for body mass index (WHRadjBMI). Genome-wide association studies (GWAS) identified 346 loci near 495 genes associated with WHRadjBMI. Most of these genes have unknown roles in fat distribution, but many are expressed and putatively act in adipose tissue. We aimed to identify novel sex- and depot-specific drivers of WHRadjBMI using a systems genetics approach. METHODS: We used two independent cohorts of adipose tissue gene expression with 362 - 444 males and 147 - 219 females, primarily of European ancestry. We constructed sex- and depot- specific Bayesian networks to model the gene-gene interactions from 8,492 adipose tissue genes. Key driver analysis identified genes that, in silico and putatively in vitro, regulate many others, including the 495 WHRadjBMI GWAS genes. Key driver gene function was determined by perturbing their expression in human subcutaneous pre-adipocytes using lenti-virus or siRNA. RESULTS: 51 - 119 key drivers in each network were replicated in both cohorts. We used single-cell expression data to select replicated key drivers expressed in adipocyte precursors and mature adipocytes, prioritized genes which have not been previously studied in adipose tissue, and used public human and mouse data to nominate 53 novel key driver genes (10 - 21 from each network) that may regulate fat distribution by altering adipocyte function. In other cell types, 23 of these genes are found in crucial adipocyte pathways: Wnt signaling or mitochondrial function. We selected seven genes whose expression is highly correlated with WHRadjBMI to further study their effects on adipogenesis/Wnt signaling (ANAPC2, PSME3, RSPO1, TYRO3) or mitochondrial function (C1QTNF3, MIGA1, PSME3, UBR1).Adipogenesis was inhibited in cells overexpressing ANAPC2 and RSPO1 compared to controls. RSPO1 results are consistent with a positive correlation between gene expression in the subcutaneous depot and WHRadjBMI, therefore lower relative storage in the subcutaneous depot. RSPO1 inhibited adipogenesis by increasing ß-catenin activation and Wnt-related transcription, thus repressing PPARG and CEBPA. PSME3 overexpression led to more adipogenesis than controls. In differentiated adipocytes, MIGA1 and UBR1 downregulation led to mitochondrial dysfunction, with lower oxygen consumption than controls; MIGA1 knockdown also lowered UCP1 expression. SUMMARY: ANAPC2, MIGA1, PSME3, RSPO1, and UBR1 affect adipocyte function and may drive body fat distribution.

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.

SEMA7a primes integrin α5ß1 engagement instructing fibroblast mechanotransduction, phenotype and transcriptional programming.

Integrins are cellular receptors that bind the extracellular matrix (ECM) and facilitate the transduction of biochemical and biophysical microenvironment cues into cellular responses. Upon engaging the ECM, integrin heterodimers must rapidly strengthen their binding with the ECM, resulting in the assembly of force-resistant and force-sensitive integrin associated complexes (IACs). The IACs constitute an essential apparatus for downstream signaling and fibroblast phenotypes. During wound healing, integrin signaling is essential for fibroblast motility, proliferation, ECM reorganization and, ultimately, restoration of tissue homeostasis. Semaphorin 7A (SEMA7a) has been previously implicated in post-injury inflammation and tissue fibrosis, yet little is known about SEMA7a's role in directing stromal cell, particularly fibroblast, behaviors. We demonstrate that SEMA7a regulates integrin signaling through cis-coupling with active integrin α5ß1 on the plasma membrane, enabling rapid integrin adhesion strengthening to fibronectin (Fn) and normal downstream mechanotransduction. This molecular function of SEMA7a potently regulates fibroblast adhesive, cytoskeletal, and migratory phenotype with strong evidence of downstream alterations in chromatin structure resulting in global transcriptomic reprogramming such that loss of SEMA7a expression is sufficient to impair the normal migratory and ECM assembly phenotype of fibroblasts resulting in significantly delayed tissue repair in vivo.

Predicting mechanisms of action at genetic loci associated with discordant effects on type 2 diabetes and abdominal fat accumulation.

Obesity is a major risk factor for cardiovascular disease, stroke, and type 2 diabetes (T2D). Excessive accumulation of fat in the abdomen further increases T2D risk. Abdominal obesity is measured by calculating the ratio of waist-to-hip circumference adjusted for the body-mass index (WHRadjBMI), a trait with a significant genetic inheritance. Genetic loci associated with WHRadjBMI identified in genome-wide association studies are predicted to act through adipose tissues, but many of the exact molecular mechanisms underlying fat distribution and its consequences for T2D risk are poorly understood. Further, mechanisms that uncouple the genetic inheritance of abdominal obesity from T2D risk have not yet been described. Here we utilize multi-omic data to predict mechanisms of action at loci associated with discordant effects on abdominal obesity and T2D risk. We find six genetic signals in five loci associated with protection from T2D but also with increased abdominal obesity. We predict the tissues of action at these discordant loci and the likely effector Genes (eGenes) at three discordant loci, from which we predict significant involvement of adipose biology. We then evaluate the relationship between adipose gene expression of eGenes with adipogenesis, obesity, and diabetic physiological phenotypes. By integrating these analyses with prior literature, we propose models that resolve the discordant associations at two of the five loci. While experimental validation is required to validate predictions, these hypotheses provide potential mechanisms underlying T2D risk stratification within abdominal obesity.

Network Preservation Analysis Reveals Dysregulated Metabolic Pathways in Human Vascular Smooth Muscle Cell Phenotypic Switching.

BACKGROUND: Vascular smooth muscle cells are key players involved in atherosclerosis, the underlying cause of coronary artery disease. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. An in-depth characterization of their gene regulatory networks can help better understand how their dysfunction may impact disease progression. METHODS: We conducted a gene expression network preservation analysis in aortic smooth muscle cells isolated from 151 multiethnic heart transplant donors cultured under quiescent or proliferative conditions. RESULTS: We identified 86 groups of coexpressed genes (modules) across the 2 conditions and focused on the 18 modules that are least preserved between the phenotypic conditions. Three of these modules were significantly enriched for genes belonging to proliferation, migration, cell adhesion, and cell differentiation pathways, characteristic of phenotypically modulated proliferative vascular smooth muscle cells. The majority of the modules, however, were enriched for metabolic pathways consisting of both nitrogen-related and glycolysis-related processes. Therefore, we explored correlations between nitrogen metabolism-related genes and coronary artery disease-associated genes and found significant correlations, suggesting the involvement of the nitrogen metabolism pathway in coronary artery disease pathogenesis. We also created gene regulatory networks enriched for genes in glycolysis and predicted key regulatory genes driving glycolysis dysregulation. CONCLUSIONS: Our work suggests that dysregulation of vascular smooth muscle cell metabolism participates in phenotypic transitioning, which may contribute to disease progression, and suggests that AMT (aminomethyltransferase) and MPI (mannose phosphate isomerase) may play an important role in regulating nitrogen and glycolysis-related metabolism in smooth muscle cells.

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.

Female gene networks are expressed in myofibroblast-like smooth muscle cells in vulnerable atherosclerotic plaques.

Women presenting with coronary artery disease (CAD) 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. Here, we show sex-stratified gene regulatory networks (GRNs) from human carotid atherosclerotic tissue. Prioritization of these networks identified two main SMC GRNs in late-stage atherosclerosis. Single-cell RNA-sequencing mapped these GRNs to two SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like GRN was mostly expressed in plaques that were vulnerable in females. Finally, mice orthologs of the female myofibroblast-like genes showed retained expression in advanced plaques from female mice but were downregulated in male mice during atherosclerosis progression. Female atherosclerosis is driven by GRNs that promote a fibrous vulnerable plaque rich in myofibroblast-like SMCs.

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.

Multi-omic integration reveals cell-type-specific regulatory networks of insulin resistance in distinct ancestry populations.

Our knowledge of the cell-type-specific mechanisms of insulin resistance remains limited. To dissect the cell-type-specific molecular signatures of insulin resistance, we performed a multiscale gene network analysis of adipose and muscle tissues in African and European ancestry populations. In adipose tissues, a comparative analysis revealed ethnically conserved cell-type signatures and two adipocyte subtype-enriched modules with opposite insulin sensitivity responses. The modules enriched for adipose stem and progenitor cells as well as immune cells showed negative correlations with insulin sensitivity. In muscle tissues, the modules enriched for stem cells and fibro-adipogenic progenitors responded to insulin sensitivity oppositely. The adipocyte and muscle fiber-enriched modules shared cellular-respiration-related genes but had tissue-specific rearrangements of gene regulations in response to insulin sensitivity. Integration of the gene co-expression and causal networks further pinpointed key drivers of insulin resistance. Together, this study revealed the cell-type-specific transcriptomic networks and signaling maps underlying insulin resistance in major glucose-responsive tissues. A record of this paper's transparent peer review process is included in the supplemental information.

Genetic regulation of circular RNA expression in human aortic smooth muscle cells and vascular traits.

Circular RNAs (circRNAs) are a class of non-coding RNAs that have cell-type-specific expression and are relevant in cardiovascular disease. Aortic smooth muscle cells (SMCs) play a crucial role in cardiovascular disease. In this study, we employed a systems genetics approach to identify SMC circRNA transcripts and their relevance in cardiovascular traits across the genome. We quantified circRNA expression across 151 quiescent and proliferative human aortic SMCs from donors of various genetic ancestries. We identified 1,589 expressed circRNAs. Between quiescent and proliferative SMCs, we identified 173 differentially expressed circRNAs. To characterize the genetic regulation of circRNA expression, we associated the genotypes of 6.3 million single nucleotide polymorphisms (SNPs) with circRNA abundance and found 96 circRNAs that were associated with genetic loci. Three SNPs were associated with circRNA expression in proliferative SMCs but not quiescent SMCs. We identified six SNPs that had distinct association directions with circRNA isoforms from the same gene. Lastly, to identify the relevance of circRNAs in cardiovascular disease, we overlapped genetic loci associated with circRNA expression with vascular disease-related genome-wide association studies loci. We identified 14 blood pressure, one myocardial infarction, and three coronary artery disease loci, which were associated with a circRNA transcript but not an mRNA transcript. Overall, our results provide insight into the genetic basis of vascular disease traits mediated by circRNA expression.

Intersecting single-cell transcriptomics and genome-wide association studies identifies crucial cell populations and candidate genes for atherosclerosis.

AIMS: Genome-wide association studies (GWASs) have discovered hundreds of common genetic variants for atherosclerotic disease and cardiovascular risk factors. The translation of susceptibility loci into biological mechanisms and targets for drug discovery remains challenging. Intersecting genetic and gene expression data has led to the identification of candidate genes. However, previously studied tissues are often non-diseased and heterogeneous in cell composition, hindering accurate candidate prioritization. Therefore, we analysed single-cell transcriptomics from atherosclerotic plaques for cell-type-specific expression to identify atherosclerosis-associated candidate gene-cell pairs. METHODS AND RESULTS: We applied gene-based analyses using GWAS summary statistics from 46 atherosclerotic and cardiovascular disease, risk factors, and other traits. We then intersected these candidates with single-cell RNA sequencing (scRNA-seq) data to identify genes specific for individual cell (sub)populations in atherosclerotic plaques. The coronary artery disease (CAD) loci demonstrated a prominent signal in plaque smooth muscle cells (SMCs) (SKI, KANK2, and SORT1) P-adj. = 0.0012, and endothelial cells (ECs) (SLC44A1, ATP2B1) P-adj. = 0.0011. Finally, we used liver-derived scRNA-seq data and showed hepatocyte-specific enrichment of genes involved in serum lipid levels. CONCLUSION: We discovered novel and known gene-cell pairs pointing to new biological mechanisms of atherosclerotic disease. We highlight that loci associated with CAD reveal prominent association levels in mainly plaque SMC and EC populations. We present an intuitive single-cell transcriptomics-driven workflow rooted in human large-scale genetic studies to identify putative candidate genes and affected cells associated with cardiovascular traits. Collectively, our workflow allows for the identification of cell-specific targets relevant for atherosclerosis and can be universally applied to other complex genetic diseases and traits.

Adipocyte-Specific Modulation of KLF14 Expression in Mice Leads to Sex-Dependent Impacts on Adiposity and Lipid Metabolism.

Genome-wide association studies identified single nucleotide polymorphisms on chromosome 7 upstream of KLF14 to be associated with metabolic syndrome traits and increased risk for type 2 diabetes (T2D). The associations were more significant in women than in men. The risk allele carriers expressed lower levels of the transcription factor KLF14 in adipose tissues than nonrisk allele carriers. To investigate how adipocyte KLF14 regulates metabolic traits in a sex-dependent manner, we characterized high-fat diet-fed male and female mice with adipocyte-specific Klf14 deletion or overexpression. Klf14 deletion resulted in increased fat mass in female mice and decreased fat mass in male mice. Female Klf14-deficient mice had overall smaller adipocytes in subcutaneous fat depots but larger adipocytes in parametrial depots, indicating a shift in lipid storage from subcutaneous to visceral fat depots. They had reduced metabolic rates and increased respiratory exchange ratios consistent with increased use of carbohydrates as an energy source. Fasting- and isoproterenol-induced adipocyte lipolysis was defective in female Klf14-deficient mice, and concomitantly, adipocyte triglycerides lipase mRNA levels were downregulated. Female Klf14-deficient mice cleared blood triglyceride and nonesterified fatty acid less efficiently than wild-type. Finally, adipocyte-specific overexpression of Klf14 resulted in lower total body fat in female but not male mice. Taken together, consistent with human studies, adipocyte KLF14 deficiency in female but not in male mice causes increased adiposity and redistribution of lipid storage from subcutaneous to visceral adipose tissues. Increasing KLF14 abundance in adipocytes of females with obesity and T2D may provide a novel treatment option to alleviate metabolic abnormalities.

Transcriptomic-based clustering of human atherosclerotic plaques identifies subgroups with different underlying biology and clinical presentation.

Histopathological studies have revealed key processes of atherosclerotic plaque thrombosis. However, the diversity and complexity of lesion types highlight the need for improved sub-phenotyping. Here we analyze the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These plaque phenotypes were associated with clinical presentation and showed differences in cellular compositions. Validation in coronary segments showed that the molecular signature of these plaques was linked to coronary ischemia. One of the plaque types with the most severe clinical symptoms pointed to both inflammatory and fibrotic cell lineages. Further, we did a preliminary analysis of potential circulating biomarkers that mark the different plaques phenotypes. In conclusion, the definition of the plaque at risk for a thrombotic event can be fine-tuned by in-depth transcriptomic-based phenotyping. These differential plaque phenotypes prove clinically relevant for both carotid and coronary artery plaques and point to distinct underlying biology of symptomatic lesions.

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.