Correlation of computed tomography with carotid plaque transcriptomes associates calcification with lesion-stabilization

Background and aims: Unstable carotid atherosclerosis causes stroke, but methods to identify patients and lesions at risk are lacking. We recently found enrichment of genes associated with calcification in carotid plaques from asymptomatic patients. Here, we hypothesized that calcification represents a stabilising feature of plaques and investigated how macro-calcification, as estimated by computed tomography (CT), correlates with gene expression profiles in lesions. Methods: Plaque calcification was measured in pre-operative CT angiographies. Plaques were sorted into high- and lowcalcified, profiled with microarrays, followed by bioinformatic analyses. Immunohistochemistry and qPCR were performed to evaluate the findings in plaques and arteries with medial calcification from chronic kidney disease patients. Results: Smooth muscle cell (SMC) markers were upregulated in high-calcified plaques and calcified plaques from symptomatic patients, whereas macrophage markers were downregulated. The most enriched processes in high-calcified plaques were related to SMCs and extracellular matrix (ECM) organization, while inflammation, lipid transport and chemokine signaling were repressed. These findings were confirmed in arteries with high medial calcification. Proteoglycan 4 (PRG4) was identified as the most upregulated gene in association with plaque calcification and found in the ECM, SMA+ and CD68+/TRAP + cells. Conclusions: Macro-calcification in carotid lesions correlated with a transcriptional profile typical for stable plaques, with altered SMC phenotype and ECM composition and repressed inflammation. PRG4, previously not described in atherosclerosis, was enriched in the calcified ECM and localized to activated macrophages and smooth muscle-like cells. This study strengthens the notion that assessment of calcification may aid evaluation of plaque phenotype and stroke risk.The European Union’s Horizon 2020/Marie Sklodowska-Curie grant agreement No 722609 (INTRICARE);Swedish Heart and Lung FoundationSwedish Research Council (K2009-65X-2233-01-3, K2013- 65X-06816-30-4, 349-2007-8703)Uppdrag Besegra Stroke (P581/ 2011-123)Stockholm County Council (ALF2011-0260, ALF-2011- 0279)Swedish Society for Medical ResearchTore Nilsson’s FoundationMagnus Bergvall’s FoundationKarolinska Institutet FoundationEuropean Commission (722609)Publishe

Altered metabolism distinguishes high-risk from stable carotid atherosclerotic plaques.

Aims Identification and treatment of the rupture prone atherosclerotic plaque remains a challenge for reducing the bur- den of cardiovascular disease. The interconnection of metabolic and inflammatory processes in rupture prone plaques is poorly understood. Herein, we investigate associations between metabolite profiles, inflammatory mediators and vulnerability in carotid atherosclerotic plaques.Methods and results We collected 159 carotid plaques from patients undergoing endarterectomy and measured 165 different metabolites in a targeted metabolomics approach. We identified a metabolite profile in carotid plaques that associated with histologically evaluated vulnerability and inflammatory mediators, as well as presence of symptoms in patients. The distinct metabolite profiles identified in high-risk and stable plaques were in line with different transcription levels of metabolic enzymes in the two groups, suggesting an altered metabolism in high-risk plaques. The altered metabolic signature in high-risk plaques was consistent with a change to increased glycolysis, elevated amino acid utilization and decreased fatty acid oxidation, similar to what is found in activated leucocytes and cancer cells.Conclusion These results highlight a possible key role of cellular metabolism to support inflammation and a high-risk phenotype of atherosclerotic plaques. Targeting the metabolism of atherosclerotic plaques with novel metabolic radiotracers or inhibitors might therefore be valid future approaches to identify and treat the high-risk atherosclerotic plaque

Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus.

Recent genome-wide association studies (GWAS) have identified multiple new loci which appear to alter coronary artery disease (CAD) risk via arterial wall-specific mechanisms. One of the annotated genes encodes LMOD1 (Leiomodin 1), a member of the actin filament nucleator family that is highly enriched in smooth muscle-containing tissues such as the artery wall. However, it is still unknown whether LMOD1 is the causal gene at this locus and also how the associated variants alter LMOD1 expression/function and CAD risk. Using epigenomic profiling we recently identified a non-coding regulatory variant, rs34091558, which is in tight linkage disequilibrium (LD) with the lead CAD GWAS variant, rs2820315. Herein we demonstrate through expression quantitative trait loci (eQTL) and statistical fine-mapping in GTEx, STARNET, and human coronary artery smooth muscle cell (HCASMC) datasets, rs34091558 is the top regulatory variant for LMOD1 in vascular tissues. Position weight matrix (PWM) analyses identify the protective allele rs34091558-TA to form a conserved Forkhead box O3 (FOXO3) binding motif, which is disrupted by the risk allele rs34091558-A. FOXO3 chromatin immunoprecipitation and reporter assays show reduced FOXO3 binding and LMOD1 transcriptional activity by the risk allele, consistent with effects of FOXO3 downregulation on LMOD1. LMOD1 knockdown results in increased proliferation and migration and decreased cell contraction in HCASMC, and immunostaining in atherosclerotic lesions in the SMC lineage tracing reporter mouse support a key role for LMOD1 in maintaining the differentiated SMC phenotype. These results provide compelling functional evidence that genetic variation is associated with dysregulated LMOD1 expression/function in SMCs, together contributing to the heritable risk for CAD

<i>TCF21</i> and the environmental sensor aryl-hydrocarbon receptor cooperate to activate a pro-inflammatory gene expression program in coronary artery smooth muscle cells

<div><p>Both environmental factors and genetic loci have been associated with coronary artery disease (CAD), however gene-gene and gene-environment interactions that might identify molecular mechanisms of risk are not easily studied by human genetic approaches. We have previously identified the transcription factor <i>TCF21</i> as the causal CAD gene at 6q23.2 and characterized its downstream transcriptional network that is enriched for CAD GWAS genes. Here we investigate the hypothesis that TCF21 interacts with a downstream target gene, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (e.g., tobacco smoke). Perturbation of <i>TCF21</i> expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of <i>AHR</i>, its heterodimerization partner <i>ARNT</i>, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including <i>IL1A</i>, <i>MMP1</i>, and <i>CYP1A1</i>. TCF21 was shown to bind in <i>AHR</i>, <i>ARNT</i> and downstream target gene loci, and co-localization was noted for AHR-ARNT and TCF21 binding sites genome-wide in regions of HCASMC open chromatin. These regions of co-localization were found to be enriched for GWAS signals associated with cardio-metabolic as well as chronic inflammatory disease phenotypes. Finally, we show that similar to TCF21, AHR gene expression is increased in atherosclerotic lesions in mice in vivo using laser capture microdissection, and AHR protein is localized in human carotid atherosclerotic lesions where it is associated with protein kinases with a critical role in innate immune response. These data suggest that TCF21 can cooperate with AHR to activate an inflammatory gene expression program that is exacerbated by environmental stimuli, and may contribute to the overall risk for CAD.</p></div

Oncostatin M reduces atherosclerosis development in APOE3Leiden.CETP mice and is associated with increased survival probability in humans

Objective Previous studies indicate a role for Oncostatin M (OSM) in atherosclerosis and other chronic inflammatory diseases for which inhibitory antibodies are in development. However, to date no intervention studies with OSM have been performed, and its relation to coronary heart disease (CHD) has not been studied. Approach and results Gene expression analysis on human normal arteries (n = 10) and late stage/advanced carotid atherosclerotic arteries (n = 127) and in situ hybridization on early human plaques (n = 9) showed that OSM, and its receptors, OSM receptor (OSMR) and Leukemia Inhibitory Factor Receptor (LIFR) are expressed in normal arteries and atherosclerotic plaques. Chronic OSM administration in APOE*3Leiden.CETP mice (n = 15/group) increased plasma E-selectin levels and monocyte adhesion to the activated endothelium independently of cholesterol but reduced the amount of inflammatory Ly-6CHigh monocytes and atherosclerotic lesion size and severity. Using aptamer-based proteomics profiling assays high circulating OSM levels were shown to correlate with post incident CHD survival probability in the AGES-Reykjavik study (n = 5457). Conclusions Chronic OSM administration in APOE*3Leiden.CETP mice reduced atherosclerosis development. In line, higher serum OSM levels were correlated with improved post incident CHD survival probability in patients, suggesting a protective cardiovascular effect

Integrative studies implicate matrix metalloproteinase-12 as a culprit gene for large-artery atherosclerotic stroke

Background. Ischaemic stroke and coronary heart disease are important contributors to the global disease burden and share atherosclerosis as the main underlying cause. Recent evidence from a genome-wide association study (GWAS) suggested that single nucleotide polymorphisms (SNP) near the MMP12 gene at chromosome 11q22.3 were associated with large-vessel ischaemic stroke. Here, we evaluated and extended these results by examining the relationship between MMP12 and atherosclerosis in clinical and experimental studies. Methods and Results. Plasma concentrations of MMP12 were measured at baseline in 3394 subjects with high-risk for cardiovascular disease (CVD) using the Olink ProSeek CVD I array. The plasma MMP12 concentration showed association with incident cardiovascular and cerebrovascular events (130 and 67 events, respectively, over 36 months) and carotid intima-media thickness progression (P = 3.6 x 10(-5)). A GWAS of plasma MMP12 concentrations revealed that SNPs rs499459, rs613084 and rs1892971 at chr11q22.3 were independently associated with plasma MMP12 (P <5 x 10(-8)). The lead SNPs showed associations with mRNA levels of MMP12 and adjacent MMPs in atherosclerotic plaques. MMP12 transcriptomic and proteomic levels were strongly significantly increased in carotid plaques compared with control arterial tissue and in plaques from symptomatic versus asymptomatic patients. By combining immunohistochemistry and proximity ligation assay, we demonstrated that MMP12 localizes to CD68 + macrophages and interacts with elastin in plaques. MMP12 silencing in human THP-1-derived macrophages resulted in reduced macrophage migration. Conclusions. Our study supports the notion that MMP12 is implicated in large-artery atherosclerotic stroke, functionally by enhancing elastin degradation and macrophage invasion in plaques

Schematic representation of AHR-TCF21 interactions that may modulate the effect of environmental stimuli on the progression of inflammation and atherosclerotic plaque formation.

<p>Environmental toxins, including dioxin and tobacco, as well as endogenous activators such as ox-LDL activate the AHR pathway, leading to increased inflammatory burden in the plaque. TCF21 can further increase the burden by increasing AHR expression as well as interacting with AHR at its downstream genes.</p

AHR expression in HCASMC is mediated by an eQTL that modulates <i>TCF21</i> binding.

<p>(a) The downstream region of the <i>AHR</i> gene on chr7 (located on >20kb distance from the transcription end site) contains a strong open chromatin region and enhancer in HCASMC marked by ATAC-Seq, H3K27ac tracks and binding of AP1 transcription factors, JUN and JUND, as well as TCF21. Enhanced view shows that SNP rs10265174 directly overlaps the ATAC-Seq open chromatin, H3K27ac enhancer mark, JUN and JUND ChIP-Seq peaks, and is within a broad TCF21 ChIP-Seq peak. (b) The LocusZoom plot shows <i>AHR</i> gene eQTL distribution in HCASMC across 1Mb (16.9–17.9Mb) on chr7 encompassing <i>AHR</i> gene, with SNP rs10265174 being the top eQTL in the locus. (c) rs10265174 alters the position weight matrix scores for AP1 and TCF4 transcription factors (data from Haploreg). (d) TCF21 ChIP-qPCR shows enrichment at the <i>AHR</i> upstream genomic region compared to IgG control (p<0.05).</p

<i>TCF21</i> overexpression perturbs expression of chronic inflammatory genes and genes linked to vascular disease pathophysiology.

<p><b>(</b>a). Representation of GO categories that are significantly altered by the <i>TCF21</i> overexpression. Red text indicates immune related GO terms, black text indicates other categories. CTRL, control cell growth conditions; <i>TCF21</i>, <i>TCF21</i> over-expression. (b) Selected DAVID GO ontology analysis restricted to genes that show significant up- or down- regulation with <i>TCF21</i> overexpression in HCASMC. Upregulated GO clusters colored in blue, downregulated in red. (c) Co-expression modules of <i>TCF21</i> and <i>AHR-ARNT</i> show a high degree of connectivity. <i>TCF21</i> and <i>AHR</i> co-expression modules were defined using 4164 human microarray datasets through GeneFriends and visualized with Cytoscape. A few representative genes are identified by gene symbol. (d) Co-expression network of 77 CAD GWAS related genes was built using GeneFriends:Microarray, and visualized with Cytoscape. Transcription modules identify three primary clusters of CAD GWAS genes, with <i>TCF21</i> and <i>AHR</i> appearing in the same cluster. Genes <i>PDGFD</i>, <i>SMAD3</i> and <i>COL4A1</i> also appear in this cluster. A second cluster is composed primarily of lipid-related genes, such as APOA1, APOA5, APOB, LPA, etc.</p

Endogenous regulation of the AHR pathway in SMC, and in vivo expression of <i>AHR</i> in atherosclerotic vascular disease.

<p>(a) <i>IL1A</i> mRNA levels are increased by dioxin and ox-LDL, and co-treatment with αNF, an AHR pathway antagonist, reverses these effects. (b) <i>AHR</i> mRNA levels are elevated in ligated carotid arteries with ruptured plaques compared to stable plaques. (c) Laser capture microdissection shows increased <i>AHR</i> mRNA levels to be located in the plaque region of the atherosclerotic aortic root. (d) <i>AHR</i> mRNA levels are elevated in atherosclerotic human carotid arteries compared to normal arteries. (e) In vivo protein expression patterns in human carotid artery plaque tissues. AHR-TCF21 unique interactors from the BIOGRID protein-protein interaction database display clustering patterns in mass-spectrometry and high resolution isoelectric focusing proteomic data from human carotid artery plaques. Proteomic datasets were constructed from patients with asymptomatic (10) and symptomatic (10) carotid stenoses. Mass-spec and high resolution isoelectric focusing yielded 8–9000 recovered proteins. Hierarchical clustering revealed AHR to be located in a cluster of genes that include immune related genes, transcription factors, and cell cycle regulated genes, such as IRAK4, SP1, and XPO1, respectively.</p