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Transcriptional and epigenetic regulation of macrophages in atherosclerosis (ID 1402)
Genetic Regulation of Vascular Smooth Muscle Cell Function (ID 1403)
O050 - SINGLE-CELL AND SPATIAL CHARACTERIZATION OF ISCHEMIC HEART DISEASE AND HEART FAILURE FROM EX-VIVO BIOPSIES REVEALS INFLAMMATORY AND VASCULAR CHANGES IN THE RIGHT ATRIUM. (ID 923)
Abstract
Background and Aims
Ischemic heart disease is the leading cause of death worldwide, leads to 9 million deaths yearly, and lacks an effective cure, thus requiring deeper understanding of its underlying mechanisms. Notably, the consequential tissue and electrical remodeling of the right atrium can have major implications with potential arrhythmias and cellular changes reflecting the state of the heart as a whole. However, the right atrium has not been included in recent genomics dissections of cardiac diseases. In this study, we aim at exploring potential transcriptional and cellular changes occurring with heart diseases in this tissue.
Methods
We use snRNA-seq from ex-vivo human right atrium biopsies, from open-heart surgeries (49 samples). In addition we profile pericardial fluid from paired patients (20 samples). We explore ischemic heart failure and heart disease, including myocardial infarction. We perform two different spatial transcriptomics assays, 1. whole transcriptome exploration and 2. novel high (sub-cellular) resolution analysis.
Results
We show major changes in gene expression, particularly pro-inflammatory signals associated with the microvasculature. We are able to define vascular endothelial and immune subtypes at the highest resolution, with markers validated by spatial assays, enabling us to link transcriptional changes to specific populations and associate them with genetic risk loci.
Conclusions
Our study provides a rich depiction of cellular identity, with a high resolution in terms of both subtypes and conditions observed. It paves the way for further dissection of the human atrium in disease context for validation of potentially actionable targets for therapies and offers a roadmap for further single cell-level dissections.
O051 - INTEGRATION OF GENETIC AND CLINICAL DATA, WITH SINGLE-CELL TRANSCRIPTOMICS OF ATHEROSCLEROTIC PLAQUES IDENTIFIES NOVEL SMOOTH MUSCLE CELL GENES (ID 339)
Abstract
Background and Aims
Smooth muscle cells (SMCs) have been causally implicated with disease processes in atherosclerosis. Single-cell RNA sequencing (scRNAseq) studies of atherosclerotic plaques have identified the presence of transitional mesenchymal cell (MC) populations. Here, we aimed to associate cell fractions from plaques to patient clinical and genetic parameters to identify novel MC-specific and symptom-specific signatures.
Methods
Deconvolution analysis was performed on bulk microarray from carotid plaques in Biobank of Karolinska Endarterectomies (BiKE, n=127) using public scRNAseq data from coronary plaques (n=5). Cell-fraction QTL (cfQTL), and plaque gene expression QTL (plaque eQTL) were calculated for a catalogue of CAD-associated GWAS loci to obtain MC-specific and symptom-specific SNPs. The function of the gene affected by the top SNP was studied in vitro using primary human carotid SMCs.
Results
Deconvolution analyses revealed that SMC cell fractions were significantly reduced in plaques from symptomatic patients. cfQTL and plaque eQTL analyses generated 84 eQTLs highly relevant for MC function. The most significant eQTL was identified as a SNP located at the regulatory region of gene ARNTL. This SNP was associated with expression levels of ARNTL specifically in plaques from symptomatic patients. Silencing ARNTL in SMCs in vitro led to inhibition of proliferation (2-way ANOVA, p < 0.01), increase in contractility (student t-test, p < 0.05), induction of senescence measured by b-galactosidase assay (student t-test, p < 0.05) and senescence markers (student t-test, p < 0.05).
Conclusions
This study identified several novel SNPs that may influence SMC function in plaques and provide insights into novel MC-specific genetic links to symptomatic atherosclerosis.
O052 - UNCOVERING IMPACT OF OBESITY ON VASCULATURE IN ORGAN- AND CELL-SUBTYPE SPECIFIC MANNER (ID 151)
Abstract
Background and Aims
Obesity promotes diverse pathologies, including atherosclerosis and dementia, which frequently involve vascular defects and endothelial cell (EC) dysfunction. Each organ has distinct EC subtypes, but whether ECs are differentially affected by obesity is unknown. Here, we aim to identify organ-specific vulnerabilities of endothelial cells in mouse obesity model.
Methods
We utilize scRNA-seq to analyse transcriptomes of ~375,000 ECs from seven organs in male mice at progressive stages of obesity induced by Western diet. We confirm our findings with orthogonal approaches including immunofluorescent stainings, tracing experiments, and in situ RNA hybridization.
Results
We reveal unique organ- and EC-subtype specific molecular changes driven by obesity, including lipid handling, metabolic pathways, AP1 transcription factor and inflammatory signaling. The transcriptomic aberrations worsen with sustained obesity and are only partially mitigated by dietary intervention and weight loss. By switching obese animals onto a chow diet, we uncover distinct obesity-driven changes in ECs that are responsive or resistant to weight loss. For example, dietary intervention substantially attenuates dysregulation of liver and adipose tissue, but not of kidney EC transcriptomes. Through integration with human GWAS data, we further identify a subset of vascular disease risk genes that are induced by obesity.
Conclusions
Our work catalogues the impact of obesity on the endothelium, and provides a useful resource for further investigation of potential organ-specific vascular therapeutic targets. The data generated in this study are available through an interactive website - https://obesity-ecatlas.helmholtz-muenchen.de.
O053 - RNA-BINDING PROTEIN HUR CONTROLS VASCULAR ENDOTHELIAL CELL INFLAMMATORY RESPONSES TO TUMOR NECROSIS FACTOR-A AND IS ASSOCIATED WITH ATHEROSCLEROSIS PROGRESSION IN HUMANS (ID 162)
Abstract
Background and Aims
RNA-binding protein HuR may control the fate of pro-inflammatory and pro-thrombotic genes in human atherosclerosis.
Methods
Single-cell RNA-sequencing (scRNA)-data were analyzed to compare cell-specific expression profiles of HuR and its target genes in murine and human atherosclerotic vascular disease. HuR individual-nucleotide cross-linking and immunoprecipitation (iCLiP), small interfering RNA-mediated HuR silencing, HuR overexpression and RNA stability assays were used to determine HuR-mediated regulation of pro-atherosclerotic genes. Expression levels of HuR were measured in peripheral blood mononuclear cells derived from 718 individuals at risk for cardiometabolic disease or with established chronic coronary syndromes (CCS) and followed for major adverse cardiovascular events (MACE).
Results
scRNA-data revealed increased HuR expression levels in vascular endothelium and infiltrated immune cells. HuR iCLiP experiments revealed HuR binding sites in the 3’ untranslated region of several genes in endothelial cells. Silencing of HuR reduces, overexpression of HuR induces the expression of targeted genes (P<0.05 for all). Inhibition of transcription by actinomycin D showed that endothelial HuR controls the mRNA stability of CCL2, CTSS, CXCL1, CXCL2 and SERPINE1 (P<0.05 for all). In humans, HuR mRNA expression was increased in CCS compared to non-coronary artery disease (CAD) participants (p<0.001) and independently associated with the presence of CAD (OR=2.67 for highest vs. lower HuR tertiles) after adjustment for traditional risk factors. High HuR concentrations independently associated with MACE incidence across a median follow-up period of 48 months (higher versus lowest tertile: 7.4% vs. 1.27%, log rank test P=0.009).
Conclusions
HuR controls the stability of pro-atherosclerotic targets, predicts the progression of atherosclerosis and MACE in humans.
O054 - DNMT3A CHIP-DRIVER MUTATIONS IGNITE THE BONE MARROW (ID 406)
Abstract
Background and Aims
The study aimed to identify phenotypical changes directly attributed to DNMT3A-CHIP driver mutations in blood and bone marrow myeloid cells from patients with coronary artery disease risk.
Methods
We conducted a prospective, observational study to screen CHIP-driver mutations in patients who were admitted for percutaneous coronary intervention. We established a single-cell DNA/RNA parallel sequencing method to acquire whole transcriptome and DNMT3A genotype of the same single blood monocytes from four DNMT3A mutation carriers and three matched non-carriers. We also applied such parallel sequencing method to bone marrow stem and progenitor cells from one DNMT3A hotspot mutation carrier, who underwent sternotomy.
Results
One third of patients in our cohort (53 out of 178) harbored CHIP-driver mutations with DNMT3A and TET2 accounting for 83% of the mutated CHIP genes. Middle-aged DNMT3A or TET2 carriers presented higher severity of coronary artery stenosis than the non-carriers. A total of 1268 blood monocytes were profiled with the parallel sequencing method, whereby we revealed that monocytes from DNMT3A mutation carriers upregulated pro-inflammatory markers, such as the S100A8 and S100A9 alarmins, in comparison with those from the non-carriers. Intra-individual comparison of approximately 100 mutant and 100 non-mutant monocytes of each carrier manifested an identical transcriptome profile, whereas the mutant hematopoietic stem and progenitor cells upregulated secretory cytokines, such as IL-1B and HMGB1, as opposed to their non-mutant counterparts.
Conclusions
Few bone marrow progenitor cells carrying DNMT3A-CHIP driver mutations suffice to induce proatherogenic inflammation in all monocytes –mutant and non-mutant ones alike.