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Objective— Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. Approach and Results— Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7 , and PLN expression positively correlated to typical SMC markers in plaques (Pearson r >0.6, P <0.0001) and in rat intimal hyperplasia ( r >0.8, P <0.0001). By immunohistochemistry, the proteins were expressed in SMCs in normal vessels, but largely absent in human plaques and intimal hyperplasia. Subcellularly, most proteins localized to the cytoskeleton in cultured SMCs and were regulated by active enhancer histone modification H3K27ac by chromatin immunoprecipitation-sequencing. Functionally, the genes were downregulated by PDGFB (platelet-derived growth factor beta) and IFNg (interferron gamma), exposure to shear flow stress, and oxLDL (oxidized low-density lipoprotein) loading. Genetic variants in PDLIM7, PLN , and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. Conclusions— We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation.

Background: Long noncoding RNAs (lncRNAs) are important regulators of biological processes involved in vascular tissue homeostasis and disease development. The current study assessed the functional contribution of the lncRNA Myocardial Infarction Associated Transcript ( MIAT ) to atherosclerosis and carotid artery disease. Methods: We profiled differences in RNA transcript expression in patients with advanced carotid artery atherosclerotic lesions from the Biobank of Karolinska Endarterectomies (BiKE). The lncRNA MIAT was identified as the most upregulated non-coding RNA transcript in carotid plaques compared to non-atherosclerotic control arteries, which was confirmed by quantitative real time PCR (qRT-PCR) and in situ hybridization. Results: Experimental knockdown of MIAT , utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (SMCs), but also increased their apoptosis. Mechanistically, MIAT regulated SMC proliferation via the EGR1-ELK1-ERK pathway. MIAT is further involved in SMC phenotypic transition to proinflammatory macrophage-like cells through binding to the promoter region of KLF4 and enhancing its transcription. Studies using Miat −/− and Miat −/− ApoE −/− mice as well as Yucatan LDLR −/− mini-pigs confirmed the regulatory role of this lncRNA in SMC de- and trans-differentiation and advanced atherosclerotic lesion formation. Conclusions: The lncRNA MIAT is a novel regulator of cellular processes in advanced atherosclerosis that controls proliferation, apoptosis, and phenotypic transition of SMCs as well as the pro-inflammatory properties of macrophages.

BackgroundPatients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysm. In the present study, collagen homeostasis in nondilated and dilated aorta segments from patients with BAV was studied, with normal and dilated aortas from tricuspid aortic valve (TAV) patients as reference.Methods and ResultsAscending aortas from 56 patients were used for biochemical and morphological analyses of collagen. mRNA expression was analyzed in 109 patients. Collagen turnover rates were similar in nondilated and dilated aortas of BAV patients, showing that aneurysmal formation in BAV is, in contrast to TAV, not associated with an increased collagen turnover. However, BAV in general was associated with an increased aortic collagen turnover compared with nondilated aortas of TAV patients. Importantly, the ratio of hydroxylysyl pyridinoline (HP) to lysyl pyridinoline (LP), 2 distinct forms of collagen cross‐linking, was lower in dilated aortas from patients with BAV, which suggests that BAV is associated with a defect in the posttranslational collagen modification. This suggests a deficiency at the level of lysyl hydroxylase (PLOD1), which was confirmed by mRNA and protein analyses that showed reduced PLOD1 expression but normal lysyl oxidase expression in dilated aortas from patients with BAV. This suggests that impaired collagen cross‐linking in BAV patients may be attributed to changes in the expression and/or activity of PLOD1.ConclusionsOur results demonstrate an impaired biosynthesis and posttranslational modification of collagen in aortas of patients with BAV, which may explain the increased aortic aneurysm formation in BAV patients.