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    Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs. Jacobsen Kevin,Lund Marie Bek,Shim Jeong,Gunnersen Stine,Füchtbauer Ernst-Martin,Kjolby Mads,Carramolino Laura,Bentzon Jacob Fog JCI insight Fibrous cap smooth muscle cells (SMCs) protect atherosclerotic lesions from rupturing and causing thrombosis, while other plaque SMCs may have detrimental roles in plaque development. To gain insight into recruitment of different plaque SMCs, we mapped their clonal architecture in aggregation chimeras of eGFP+Apoe-/- and Apoe-/- mouse embryos and in mice with a mosaic expression of fluorescent proteins in medial SMCs that were rendered atherosclerotic by PCSK9-induced hypercholesterolemia. Fibrous caps in aggregation chimeras were found constructed from large, endothelial-aligned layers of either eGFP+ or nonfluorescent SMCs, indicating substantial clonal expansion of a few cells. Similarly, plaques in mice with SMC-restricted Confetti expression showed oligoclonal SMC populations with little intermixing between the progeny of different medial SMCs. Phenotypes comprised both ACTA2+ SMCs in the cap and heterogeneous ACTA2- SMCs in the plaque interior, including chondrocyte-like cells and cells with intracellular lipid and crystalline material. Fibrous cap SMCs were invariably arranged in endothelium-aligned clonal sheets, confirming results in the aggregation chimeras. Analysis of the clonal structure showed that a low number of local medial SMCs partake in atherosclerosis and that single medial SMCs can produce several different SMC phenotypes in plaque. The combined results show that few medial SMCs proliferate to form the entire phenotypically heterogeneous plaque SMC population in murine atherosclerosis. 10.1172/jci.insight.95890
    Environment-Sensing Aryl Hydrocarbon Receptor Inhibits the Chondrogenic Fate of Modulated Smooth Muscle Cells in Atherosclerotic Lesions. Kim Juyong Brian,Zhao Quanyi,Nguyen Trieu,Pjanic Milos,Cheng Paul,Wirka Robert,Travisano Stanislao,Nagao Manabu,Kundu Ramendra,Quertermous Thomas Circulation BACKGROUND:Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. METHODS:We combined RNA-sequencing, chromatin immunoprecipitation followed by sequencing, assay for transposase-accessible chromatin using sequencing, and in vitro assays in human coronary artery SMCs, with single-cell RNA-sequencing, histology, and RNAscope in an SMC-specific lineage-tracing knockout mouse model of atherosclerosis to better understand the role of in vascular disease. RESULTS:Genomic studies coupled with functional assays in cultured human coronary artery SMCs revealed that modulates the human coronary artery SMC phenotype and suppresses ossification in these cells. Lineage-tracing and activity-tracing studies in the mouse aortic sinus showed that the pathway is active in modulated SMCs in the atherosclerotic lesion cap. Furthermore, single-cell RNA-sequencing studies of the SMC-specific knockout mice showed a significant increase in the proportion of modulated SMCs expressing chondrocyte markers such as and , which localized to the lesion neointima. These cells, which we term "chondromyocytes," were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMCs in the lesion cap, and increased alkaline phosphatase activity in lesions in the knockout in comparison with wild-type mice. We propose that is likely protective based on these data and inference from human genetic analyses. CONCLUSIONS:Overall, we conclude that promotes the maintenance of lesion cap integrity and diminishes the disease-related SMC-to-chondromyocyte transition in atherosclerotic tissues. 10.1161/CIRCULATIONAHA.120.045981
    The Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis. Alencar Gabriel F,Owsiany Katherine M,K Santosh,Sukhavasi Katyayani,Mocci Giuseppe,Nguyen Anh,Williams Corey M,Shamsuzzaman Sohel,Mokry Michal,Henderson Christopher A,Haskins Ryan,Baylis Richard A,Finn Aloke V,McNamara Coleen A,Zunder Eli R,Venkata Vamsidhar,Pasterkamp Gerard,Björkegren Johan,Bekiranov Stefan,Owens Gary K Circulation Rupture or erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a very limited understanding of the identity, origin, and function of many cells that make up late stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. We conducted a comprehensive single-cell RNA-seq of advanced human carotid endarterectomy samples and compared these with scRNA-seq from murine micro-dissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used ChIP-seq, bulk RNA-seq and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affects lesion pathogenesis. We provide evidence SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures and their putative target genes play an important role regulating SMC phenotypic changes. scRNA-seq revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and EC-derived cells including seven distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11, Lgals3 population with a chondrocyte-like gene signature that was markedly reduced with SMC- knockout. We observed that SMC that activate Lgals3 comprise up to 2/3 of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene, ECM-remodeling, "pioneer" cell phenotype that are the first to invest within lesions and subsequently give rise to at least 3 other SMC phenotypes within advanced lesions including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3 osteogenic cells likely to be detrimental for late stage atherosclerosis plaque pathogenesis. 10.1161/CIRCULATIONAHA.120.046672
    TNAP stimulates vascular smooth muscle cell trans-differentiation into chondrocytes through calcium deposition and BMP-2 activation: Possible implication in atherosclerotic plaque stability. Fakhry Maya,Roszkowska Monika,Briolay Anne,Bougault Carole,Guignandon Alain,Diaz-Hernandez Juan Ignacio,Diaz-Hernandez Miguel,Pikula Slawomir,Buchet René,Hamade Eva,Badran Bassam,Bessueille Laurence,Magne David Biochimica et biophysica acta. Molecular basis of disease Atherosclerotic plaque calcification varies from early, diffuse microcalcifications to a bone-like tissue formed by endochondral ossification. Recently, a paradigm has emerged suggesting that if the bone metaplasia stabilizes the plaques, microcalcifications are harmful. Tissue-nonspecific alkaline phosphatase (TNAP), an ectoenzyme necessary for mineralization by its ability to hydrolyze inorganic pyrophosphate (PP), is stimulated by inflammation in vascular smooth muscle cells (VSMCs). Our objective was to determine the role of TNAP in trans-differentiation of VSMCs and calcification. In rodent MOVAS and A7R5 VSMCs, addition of exogenous alkaline phosphatase (AP) or TNAP overexpression was sufficient to stimulate the expression of several chondrocyte markers and induce mineralization. Addition of exogenous AP to human mesenchymal stem cells cultured in pellets also stimulated chondrogenesis. Moreover, TNAP inhibition with levamisole in mouse primary chondrocytes dropped mineralization as well as the expression of chondrocyte markers. VSMCs trans-differentiated into chondrocyte-like cells, as well as primary chondrocytes, used TNAP to hydrolyze PP, and PP provoked the same effects as TNAP inhibition in primary chondrocytes. Interestingly, apatite crystals, associated or not to collagen, mimicked the effects of TNAP on VSMC trans-differentiation. AP and apatite crystals increased the expression of BMP-2 in VSMCs, and TNAP inhibition reduced BMP-2 levels in chondrocytes. Finally, the BMP-2 inhibitor noggin blocked the rise in aggrecan induced by AP in VSMCs, suggesting that TNAP induction in VSMCs triggers calcification, which stimulates chondrogenesis through BMP-2. Endochondral ossification in atherosclerotic plaques may therefore be induced by crystals, probably to confer stability to plaques with microcalcifications. 10.1016/j.bbadis.2016.12.003