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Epigenetic age estimators (clocks) are known to be predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques can be used for predicting secondary events. Here we estimated an age adjusted measure of epigenetic age, epigenetic age acceleration (EAA), using DNA methylation of human atherosclerotic plaques and of blood. EAA of plaque, but not blood, independently predicted secondary events in a 3-year follow-up (HR=1.3, p= 0.018). Plaque EAA concurred with a high metabolic epigenetic and transcriptional state in plaques. Patients with diabetes and a high body mass index had a higher plaque EAA. EAA was lower in female plaques compared to male plaques by approximately 2 years. Single-cell RNA-seq revealed mesenchymal smooth muscle cells and endothelial cells as main drivers of EAA. Plaque-specific ageing may help identify processes that explain poor health outcomes.

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.

Sex differences in endothelial cell (EC) biology may reflect intrinsic differences driven by chromosomes or sex steroid exposure and gender differences accumulated over life. We analysed EC gene expression data from boy–girl twins at birth and in non-twin adults to detect sex differences at different stages of life, and show that 14–25% of the EC transcriptome is sex-biased. By combining data from both stages of life, we identified sex differences that are present at birth and maintained throughout life, and those that are acquired over life. Promisingly, we found that genes that present with an acquired sex difference in ECs are more likely to be targets of sex steroids. Annotating both gene sets with data from multiple genome-wide association studies (GWAS) revealed that genes with an intrinsic sex difference in ECs are enriched for coronary artery disease GWAS hits. This study underscores the need for treating sex as a biological variable.

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. We hypothesized that unbiased clustering of plaques based on gene expression results in an alternative categorization of late-stage atherosclerotic lesions.We analyzed the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These novel plaque phenotypes associated with clinical presentation (pIn 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 differential underlying biology of symptomatic lesions.

RationaleLong noncoding RNAs (lncRNAs) control cardiac gene expression during heart development and disease. It is accordingly plausible for the same lncRNA to regulate both cardiac development, as well as play a role in adult heart disease progression. lncRNA regulators of early cardiomyocyte (CM) lineage commitment have been identified and characterised, however those controlling later CM specification remain unknown.ObjectivesIn this study we identified a novel lncRNA required for CM specification, maturation and function, and also discovered its suggested relevance to heart disease.Methods and ResultsWe performed single cell RNA-seq on human embryonic stem cell derived cardiomyocytes at 2, 6 and 12 weeks of differentiation. Weighted correlation network analysis (WGCNA) identified core gene modules, including lncRNAs highly abundant and uniquely expressed in the human heart. A lncRNA (we call VENTHEART, VHRT) co-expressed with cardiac maturation and ventricular-specific genes MYL2 and MYH7, as well as in adult human ventricular tissue. CRISPR-mediated excision of VHRT led to impaired CM sarcomere formation, and loss of the CM specification gene program. VHRT knockdown (KD) in hESC-CMs confirmed its regulatory role for key cardiac contraction, calcium hemostasis and heart development genes, including MYH6 and RYR2. Functional evaluation after VHRT KD using impedance-based technology and action potential recordings, proved reduced contraction amplitude and loss of the ventricular-like action potential in CM, respectively. Through an integrative analysis of genome-wide association studies (GWAS), expression quantitative trait locus (eQTL) and gene co-expression network, we found VHRT to be co-regulated with core cardiac contractile genes, and the likely source of a heart failure genetic association signal overlapping the VHRT gene locus. Finally, VHRT KD and human failing heart transcriptome comparison validates the consistent downregulation again of cardiac contractile and calcium regulatory genes (PConclusionWe conclude that VHRT lncRNA is required for proper CM specification and function. Furthermore, reduced VHRT may contribute to the development or progression of human heart disease.

Sex differences are evident in the clinical presentation and underlying histology of atherosclerotic disease with women developing more stable atherosclerotic lesions than men. It is unknown whether this is explained by sex differences in gene regulation in cellular compartments of atherosclerotic plaques. To study sex differences in gene regulation we performed genome-wide DNA methylation and transcriptomics analysis on plaques of 485 carotid endarterectomy patients (31% female). Sex-differential DNA methylation at 4,848 sites in the autosome was enriched for cell-fate commitment and developmental processes, and its deconvolution predicted more smooth muscle cells in females, as compared to more immune cells in males. RNA-sequencing of the same plaques corroborated the sex differences in DNA methylation predicted cell-types, in which genes that were higher expressed in females were enriched for TGF-beta signaling and extracellular matrix biology. In addition, female-biased genes were enriched for targeting by regulatory loci based on sex differential methylation. Lastly, by using single-cell RNA sequencing we showed that these female-biased genes are mostly expressed in smooth muscle cells, and higher expressed in smooth muscle cells from female (predominantly stable) plaques as compared to male (relatively unstable) plaques. Our approach identified female-biased genes in smooth muscle cells in fibrous atherosclerotic plaques. This points towards new mechanisms in smooth muscle cell biology of stable atherosclerotic plaques and offers new directions for research to develop new sex-specific therapeutics for atherosclerotic disease.

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.

Many pathophysiological mechanisms in human health and disease are dependent on sex. Systems biology approaches are successfully used to decipher human disease etiology, yet the effect of sex on gene network biology is mostly unknown. To address this, we used RNA-sequencing data of over 700 individuals spanning 24 tissues from the Genotype-Tissue Expression project to generate a whole-body gene co-expression map and quantified the sex differences per tissue. We found that of the 13,787 genes analyzed in 24 tissues, 29.5% of the gene co-expression is influenced by sex. For example, skeletal muscle was predominantly enriched with genes co-expressed stronger in males, whereas thyroid primarily contained genes co-expressed stronger in females. This was accompanied by consistent sex differences in pathway enrichment, including hypoxia, epithelial-to-mesenchymal transition, and inflammation over the human body. Furthermore, multi-organ analyses revealed consistent sex-dependent gene co-expression over numerous tissues which was accompanied by enrichment of transcription factor binding motifs in the promoters of these genes. Finally, we show that many sex-biased genes are associated with sex-biased diseases, such as autoimmunity and cancer, and more often the target of FDA-approved drugs than non-sexbiased genes. Our study suggests that sex affects biological gene networks by differences in gene co-expression and that attention to the effect of sex on biological responses to medical drugs is warranted.

Many pathophysiological mechanisms in human health and disease are dependent on sex. Systems biology approaches are successfully used to decipher human disease etiology, yet the effect of sex on gene network biology is mostly unknown. To address this, we used RNA-sequencing data of over 700 individuals spanning 24 tissues from the Genotype-Tissue Expression project to generate a whole-body gene activity map and quantified the sex differences per tissue. We found that of the 13,787 genes analyzed in 24 tissues, 20.1% of the gene activity is influenced by sex. For example, skeletal muscle was predominantly enriched with genes more active in males, whereas thyroid primarily contained genes more active in females. This was accompanied by consistent sex differences in pathway activity, including hypoxia, epithelial-to-mesenchymal transition, and inflammation over the human body. Furthermore, multi-organ analyses revealed consistent sex-dependent gene activity over numerous tissues which was accompanied by enrichment of transcription factor binding motifs in the promoters of these genes. Finally, we show that many sex-biased genes are known druggable targets. This emphasizes sex as a biological variable and the need to incorporate sex in systems biology studies.

For decades, the pathological definition of the vulnerable plaque led to invaluable insights into the mechanisms that underlie myocardial infarction and stroke. Beyond plaque rupture, other mechanisms, such as erosion, may elicit thrombotic events underlining the complexity and diversity of the atherosclerotic disease. Novel insights, based on single-cell transcriptomics and other "omics" methods, provide tremendous opportunities in the ongoing search for cell-specific determinants that will fine-tune the description of the thrombosis prone lesion. It coincides with an increasing awareness that knowledge on lesion characteristics, cell plasticity and clinical presentation of ischemic cardiovascular events have shifted over the past decades. This shift correlates with an observed changes of cell composition towards phenotypical stabilizing of human plaques. These stabilization features and mechanisms are directly mediated by the cells present in plaques and can be mimicked in vitro via primary plaque cells derived from human atherosclerotic tissues. In addition, the rapidly evolving of sequencing technologies identify many candidate genes and molecular mechanisms that may influence the risk of developing an atherosclerotic thrombotic event - which bring the next challenge in sharp focus: how to translate these cell-specific insights into tangible functional and translational discoveries?

Visual Abstract, Highlights • The interplay between cardiomyocytes and cardiac fibroblasts is increasingly being recognized as important in cardiac disease. • Fetal and adult cardiac fibroblasts influence their neighboring cardiomyocytes in different ways. A genome-wide comparison of the 2 reveals that they share >80% of gene transcripts. • Motif analysis of empirical regulatory elements located next to differentially expressed genes led to identification of key differential regulators of fibroblast identity. • STAT1 and PLAGL1 were identified and validated as key transcription factors to maintain the adult cardiac fibroblast phenotype. Loss of either factor led to a significant change in phenotype, including smaller cell size, apoptosis, reduced turnover, and down-regulated collagen gene expression., Summary Cardiovascular disease remains the number one global cause of death and presents as multiple phenotypes in which the interplay between cardiomyocytes and cardiac fibroblasts (CFs) has become increasingly highlighted. Fetal and adult CFs influence neighboring cardiomyocytes in different ways. Thus far, a detailed comparison between the two is lacking. Using a genome-wide approach, we identified and validated 2 crucial players for maintaining the adult primary human CF phenotype. Knockdown of these factors induced significant phenotypical changes, including senescence and reduced collagen gene expression. These may now represent novel therapeutic targets against deleterious functions of CFs in adult cardiovascular disease.

Heart failure (HF) is one of the major health threats of western societies. Women are more likely to survive than men and HF in women frequently occurs at an older age and with less ischemic etiology than men. In western populations, HFpEF has a greater prevalence in women and HFrEF in men. Risk factors for HF differ in women and men. Hypertension contributes to the risk for heart failure in women to a greater extent than in men and diabetes is a stronger risk factor for HF in women than in men. The interaction between diabetes and atherogenic and prohypertrophic mechanisms and cardiovascular risk in women has been related to sexual hormones. Acute coronary syndromes (ACS) occur 3–4 times more often in men than women below age 60, but after 75 years women represent the majority of patients. IHD seems to lead more frequently to HF in women than in men. Dilated cardiomyopathy and hypertrophic cardiomyopathy are more frequent causes of HF in men than in women.

Background and aims Women who develop preeclampsia during pregnancy are at a higher risk for developing cardiovascular disease. As platelets are affected by preeclampsia, we set out to identify whether platelets carry information in their transcriptome on cardiovascular risk in women with former preeclampsia. Methods Platelets were isolated from asymptomatic women with previous preeclampsia, who underwent screening with coronary computed tomography angiography. Platelet RNA was isolated and used to construct gene networks using an unbiased approach. Platelet gene modules assembled from the network were related to risk factors and clinical traits of these women, including coronary artery calcium scores (CACS). Results We found multiple gene modules which correlated with CACS (correlation coefficients: 0.44 to 0.59, p = 0.05 to 0.007). The genes from two clinically relevant modules were expressed at a higher level in the group with calcifications (p = 3.9 × 10−10 and 0.02) and enriched for platelet-related gene-sets such as platelet activation. The first of these modules was also enriched (ppermutation = 0.0546) for genes mapped to known coronary artery disease susceptibility loci. Additional unbiased network analyses in platelet RNA of patients with overt cardiovascular disease underlined the importance of the identified modules for disease by high preservation. (p = 1.6 × 10−9 to 1.7 × 10−47). Conclusions We found platelet RNA modules that correlated with CACS in asymptomatic women with previous preeclampsia. Whether or not platelets directly contribute to this disease trajectory, or reflect the underlying plaque substrate remains to be determined, but enrichment for coronary artery disease susceptibility genes emphasizes the importance for the disease.

Background Differences in cardiovascular diseases are evident in men and women throughout life and are mainly attributed to the presence of sex hormones and chromosomes. Epigenetic mechanisms drive the regulation of the biological processes that may lead to CVD and are possibly influenced by sex. In order to gain an overview of the status quo on sex differences in cardiovascular epigenetics, we performed a systematic review. Materials and methods A systematic search was performed on PubMed and Embase for studies mentioning cardiovascular disease, epigenetics, and anything related to sex differences. The search returned 3071 publications to be screened. Primary included publications focused on cardiovascular and epigenetics research. Subsequently, papers were assessed for including both sexes in their studies and checked for appropriate sex stratification of results. Results Two independent screeners identified 75 papers in the proper domains that had included both sexes. Only 17% (13 papers out of 75) of these publications stratified some of their data according to sex. All remaining papers focused on DNA methylation solely as an epigenetic mechanism. Of the excluded papers that included only one sex, 86% (24 out 28) studied males, while 14% (4 out of 28) studied females. Conclusion Our overview indicates that the majority of studies into cardiovascular epigenetics do not show their data stratified by sex, despite the well-known sex differences in CVD. All included and sex-stratified papers focus on DNA methylation, indicating that a lot of ground is still to gain regarding other epigenetic mechanisms, like chromatin architecture, and histone modifications. More attention to sex in epigenetic studies is warranted as such integration will advance our understanding of cardiovascular disease mechanisms in men and women. Electronic supplementary material The online version of this article (10.1186/s13293-018-0180-z) contains supplementary material, which is available to authorized users.