Johan L M Björkegren1,2,3, Hester M den Ruijter4, Robin J G Hartman4, Katie Owsiany5,6, Lijiang Ma1, Simon Koplev1, Ke Hao1,2, Lotte Slenders7, Mete Civelek8, Michal Mokry4,7, Jason C Kovacic9,10, Gerard Pasterkamp7, Gary Owens5. 1. University of Virginia-School of Medicine, Charlottesville. Department of Genetics and Genomic Sciences (L.M., S.K., K.H., J.L.M.B.), New York. Victor Chang Cardiac Research Institute, Darlinghurst, Australia. 2. Icahn Institute of Genomics and Multiscale Biology (K.H., J.L.M.B.), New York. Victor Chang Cardiac Research Institute, Darlinghurst, Australia. 3. Integrated Cardio Metabolic Centre, Department of Medicine, Karolinska Institutet, Karolinska Universitetssjukhuset, Huddinge, Sweden (J.L.M.B.). 4. Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht University, The Netherlands (R.J.G.H., M.M., H.M.d.R.). 5. Robert M. Berne Cardiovascular Research Center (K.O., G.O.), New York. Victor Chang Cardiac Research Institute, Darlinghurst, Australia. 6. Department of Biochemistry and Molecular Genetics (K.O.), New York. Victor Chang Cardiac Research Institute, Darlinghurst, Australia. 7. Central Diagnostics Laboratory, University Medical Center Utrecht, Utrecht University, The Netherlands (L.S., M.M., G.P.). 8. Center for Public Health Genomics, Department of Biomedical Engineering (M.C.), New York. Victor Chang Cardiac Research Institute, Darlinghurst, Australia. 9. Icahn School of Medicine at Mount Sinai (J.C.K.), New York. Victor Chang Cardiac Research Institute, Darlinghurst, Australia. 10. St Vincent's Clinical School, University of NSW (J.C.K.).
Abstract
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.
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.
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