Herein we employ Myh11-CreERT2 ROSA floxed STOP eYFP Apoe−/− smooth muscle cell (SMC) lineage tracing mice to show that traditional methods for detecting SMCs based on immuno-staining fail to detect > 80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages including macrophages (Mϕs), and mesenchymal stem cells (MSCs). SMC-specific conditional knockout (KO) of Krüppel-like factor 4 (KLF4) resulted in reduced numbers of SMC-derived MSC-, and Mϕ-like cells, marked reductions in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness. Results of in vivo KLF4 ChIP-Seq analyses, and studies in cultured SMC treated with cholesterol identified > 800 KLF4 target genes including many that regulate pro-inflammatory responses of SMC. Results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.

Despite decades of research, our understanding of the processes controlling late-stage atherosclerotic plaque stability remains poor. A prevailing hypothesis is that reducing inflammation may improve advanced plaque stability, as recently tested in the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial, in which post-myocardial infarction subjects were treated with an IL-1β antibody. Here, we performed intervention studies in which smooth muscle cell (SMC) lineage-tracing Apoe mice with advanced atherosclerosis were treated with anti-IL-1β or IgG control antibodies. Surprisingly, we found that IL-1β antibody treatment between 18 and 26 weeks of Western diet feeding induced a marked reduction in SMC and collagen content, but increased macrophage numbers in the fibrous cap. Moreover, although IL-1β antibody treatment had no effect on lesion size, it completely inhibited beneficial outward remodeling. We also found that SMC-specific knockout of Il1r1 (encoding IL-1 receptor type 1) resulted in smaller lesions nearly devoid of SMCs and lacking a fibrous cap, whereas macrophage-selective loss of IL-1R1 had no effect on lesion size or composition. Taken together, these results show that IL-1β has multiple beneficial effects in late-stage murine atherosclerosis, including promotion of outward remodeling and formation and maintenance of an SMC- and collagen-rich fibrous cap.

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Stable atherosclerotic plaques are characterized by a thick extracellular matrix (ECM)-rich fibrous cap populated by protective ACTA2 + myofibroblast (MF)-like cells, assumed to be almost exclusively derived from smooth muscle cells (SMC). Herein, we show that in murine and human lesions, 20 to 40% of ACTA2 + fibrous caps cells, respectively, are derived from non-SMC sources, including endothelial cells (EC) or macrophages that have undergone Endothelial-to-Mesenchymal (EndoMT) or Macrophage-to-Mesenchymal (MMT) transitions. In addition, we show that SMC-specific knockout of the platelet derived growth factor receptor beta (PDGFRB) in Apoe −/− mice fed a Western diet (WD) for 18 weeks resulted in brachiocephalic artery (BCA) lesions nearly devoid of SMC but with no changes in lesion size, remodeling, or indices of stability including percent of ACTA2 + fibrous cap cells. However, prolonged WD feeding of SMC-PDGFRB knockout mice resulted in reduced indices of stability, indicating that EndoMT and MMT-derived MFs cannot compensate indefinitely for loss of SMC-derived MFs. Using single cell and bulk RNA-seq analyses of the BCA region and in vitro models, we provide evidence that SMC to MF transitions (SMC-MFT) are induced by PDGF and TFGβ and dependent on aerobic glycolysis, while EndoMT is induced by IL1β and TGFβ. Together, we provide evidence that the ACTA2 + fibrous cap originates from a tapestry of cell types, which transition to an MF state through distinct signaling pathways that are either dependent on or associated with extensive metabolic reprogramming.