BACKGROUND AND AIMS:The role of vascular smooth muscle cells (SMCs) in atherosclerosis has evolved to indicate causal genetic links with the disease. Single cell RNA sequencing (scRNAseq) studies have identified multiple cell populations of mesenchymal origin within atherosclerotic lesions, including various SMC sub-phenotypes, but it is unknown how they relate to patient clinical parameters and genetics. Here, mesenchymal cell populations in atherosclerotic plaques were correlated with major coronary artery disease (CAD) genetic variants and functional analyses performed to identify SMC markers involved in the disease. METHODS:Bioinformatic deconvolution was done on bulk microarrays from carotid plaques in the Biobank of Karolinska Endarterectomies (BiKE, n = 125) using public plaque scRNAseq data and associated with patient clinical data and follow-up information. BiKE patients were clustered based on the deconvoluted cell fractions. Quantitative trait loci (QTLs) analyses were performed to predict the effect of CAD associated genetic variants on mesenchymal cell fractions (cfQTLs) and gene expression (eQTLs) in plaques. RESULTS:Lesions from symptomatic patients had higher fractions of Type 1 macrophages and pericytes, but lower fractions of classical and modulated SMCs compared with asymptomatic ones, particularly females. Presence of diabetes or statin treatment did not affect the cell fraction distribution. Clustering based on plaque cell fractions, revealed three patient groups, with relative differences in their stability profiles and associations to stroke, even during long-term follow-up. Several single nucleotide polymorphisms associated with plaque mesenchymal cell fractions, upstream of the circadian rhythm gene ARNTL were identified. In vitro silencing of ARNTL in human carotid SMCs increased the expression of contractile markers and attenuated cell proliferation. CONCLUSIONS:This study shows the potential of combining scRNAseq data with vertically integrated clinical, genetic, and transcriptomic data from a large biobank of human plaques, for refinement of patient vulnerability and risk prediction stratification. The study revealed novel CAD-associated variants that may be functionally linked to SMCs in atherosclerotic plaques. Specifically, variants in the ARNTL gene may influence SMC ratios and function, and its role as a regulator of SMC proliferation should be further investigated.

Intro: Vascular smooth muscle cell (VSMC) proliferation following endovascular interventions leads to restenosis, treated with drug-eluting stents. However, current drugs completely abolish vascular healing, cause thrombosis and clinical events. PCSK6 is a key protease in vascular remodeling and VSMCs activation upon vessel injury, via regulation of MMP2/14 activity. Here, we investigate whether local PCSK6 inhibition reduces VSMC proliferation and intimal hyperplasia. Methods: PCSK6 peptide inhibitor was tested in vitro on VSMCs migration, proliferation, apoptosis in comparison to rapamycin and paclitaxel. Internalization of a FITC labelled inhibitor was studied by flow cytometry and immunofluorescence. In vivo , PCSK6 inhibitor was administered at 3mg/kg IP using cationic microbubbles, followed by ultrasound-mediated local delivery in mice developing intimal hyperplasia after carotid ligation (n=8 vs 11). Carotid ligation in Tagln Cre+ /Pcsk6 fl/fl mice investigated the effect of PCSK6 specific ablation in VSMCs on intimal hyperplasia (n=7 vs 9). Carotids were collected from these and constitutive Pcsk6 -/- mice vs. controls for transcriptomic and histological analyses. Results: In vitro , PCSK6 inhibitor was internalized and inhibited both VSMC migration and proliferation (p<0.0001), reducing the expression of PCSK6, MMP2 and GDF15 . In comparison, paclitaxel showed severe VSMC toxicity, whereas rapamycin inhibited more strongly VSMC migration and proliferation than the PCSK6 inhibitor. In vivo, the inhibitor reduced intimal hyperplasia (p=0.026), again, by repressing PCSK6 and MMP2 expression in treated vs. control mice, with a favorable safety profile. Likewise, conditional VSMC Pcsk6 knockouts showed less intimal hyperplasia (p=0.053). In carotids from Pcsk6 -/- mice, cytoskeletal (Synm) and peptidase (Pgpep1l, Klk1) genes were strongly downregulated, showing its importance for vascular matrix composition. Conclusion: Our proof-of-concept translational study shows that local application of a PCSK6 inhibitor is a viable method for specifically modulating VSMC activation in intimal hyperplasia following vascular injury

Understanding the pathophysiology of unstable atherosclerosis, the underlying cause of myocardial infarction and ischemic stroke, is imperative to catalyze novel translational strategies. Our objective was to identify key mechanisms and signatures of atherosclerotic instability, by leveraging the power of multi-omics integration from a large biobank. Biobank of Karolinska Endarterectomies (BiKE), with biomaterial and data from patients with symptomatic (S) and asymptomatic (AS) carotid atherosclerosis, was the main resource in this study. Matched plaques, PBMCs, plasma sampled locally around the plaque and from the periphery in n>600 individuals, were profiled by orthogonal genomic, transcriptomic, proteomic and metabolomic technologies. Single-omics analyses were performed for differential expression, pathway and network studies in S vs. AS comparisons. Multi-omics integration was performed using a benchmarked method DIABLO, across local and peripheral disease sites and their intersection. Top targets were investigated further using scRNAseq, patient clinical and outcome data, as well as QTL analyses coupling target genetics with transcript, protein, and metabolite levels. Plaque and PBMC transcriptomics showed that inflammatory signalling, hypoxia, angiogenesis, ECM reorganisation, bone remodelling, cholesterol homeostasis, glycolysis were enriched pathways. In proteomics, chemokine signalling upregulation was found in both peripheral and local plasma, while IL17 pathway was enriched in local plasma from S patients. In plasma metabolomics, Bilirubin degradation products were downregulated, while Sphingomyelins were upregulated. Multi-omics integrations revealed coagulation, necroptosis, immune activation and cholesterol metabolism as key pathways determining instability. Extended analyses of the top analytes FABP4, ICOSLG, ANGPTL3, Bilirubin and Sphingomyelins, generated an insight into their association with survival, clinical and genetic risk in patients. Using an innovative framework, this study brings novel information about atherosclerosis vulnerability, opening avenues for drug target and biomarker discoveries, but also improving personalised treatment options and risk stratifications.

Background and Aim: A central role of vascular smooth muscle cells (SMCs) in atherosclerosis has recently evolved that suggests causal genetic links to disease processes. Single cell sequencing studies of atherosclerotic plaques have identified multiple mesenchymal transition cell populations within the plaques. Here, we correlated cell fractions from plaques to coronary artery disease (CAD) related gene polymorphisms to identify novel SMC targets and study their influence on SMC function in atherosclerosis. Methods: Deconvolution analysis was performed on bulk microarray data from carotid plaques in the Biobank of Karolinska Endarterectomies (BiKE, n=127) using single cell sequencing data from coronary plaques (n=5). CAD-associated GWAS loci associated with mesenchymal cell fractions were identified, followed by functional analyses of these genes in SMC in vitro using migration, proliferation and apoptosis assays. Results: We identified 5 mesenchymal cell-specific genetic variants associated with CAD, BiKE patient symptomatology and gene expression eQTLs in BiKE plaque tissue and GTex normal arteries. These variants were harbored in genetic loci of ARNTL, LDLR, MIA3, PAK1 and ARHGAP15 . Microarray analysis revealed increased expression of ARHGAP15 and PAK1 and decreased levels of LDLR in carotid plaques compared with normal arteries (n=127 vs. 10 respectively, student’s t-test). Immunohistochemistry demonstrated increased expression of corresponding proteins in the fibrous cap of plaques compared to normal arteries (n=5). To investigate their function in SMCs, the genes were silenced using siRNAs followed by migration, proliferation and apoptosis assays. Preliminary results indicated that silencing of MIA3, LDLR and ARNTL inhibited SMC proliferation. Conclusions: The results of this project may reveal novel SMC-specific genetic links to the disease, which may serve as therapeutic targets to be explored for improved treatment of atherosclerosis.