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Rationale: Plaque instability remains poorly understood and new therapeutic approaches to reduce plaque rupture and subsequent clinical events are of great interest. Recent studies revealed an important role of phenotypic switching of smooth muscle cells (SMC) in controlling plaque stability, including ECM (extracellular matrix) deposition. Objective: The aim of this study was to elucidate the role of hyaluronan derived from SMC–hyaluronan synthase 3 ( Has3 ), in phenotypic switching and plaque stability in an animal model of atherosclerosis. Methods and Results: A mouse line with SMC-specific deletion of Has3 and simultaneous SMC-lineage tracing ( e YFP [enhanced yellow fluorescent protein]) on an Apoe −/− background was used. Lineage tracing of SMC with e YFP revealed that SMC-specific deletion of Has3 significantly increased the number of LGALS3 + (galectin-3) transition state SMC and decreased ACTA2 + (alpha-smooth muscle actin) SMC. Notably, SMC- Has3 deletion led to significantly increased collagen deposition and maturation within the fibrous cap and the whole lesion, as evidenced by picrosirius red staining and LC-PolScope analysis. Single-cell RNA sequencing of brachiocephalic artery lesions demonstrated that the loss of SMC- Has3 enhanced the transition of SMC to a Lgals3 + , ECM-producing phenotype with elevated acute-phase response gene expression. Experiments using cultured murine aortic SMC revealed that blocking CD44 (cluster of differentiation-44), an important hyaluronan binding receptor, recapitulated the enhanced acute-phase response, and synthesis of fibrous ECM. Conclusions: These studies provide evidence that the deletion of SMC- Has3 results in an ECM-producing transition state SMC phenotype (characterized by LGALS3 + expression), likely via reduced CD44 signaling, resulting in increased collagen formation and maturation, an index consistent with increased plaque stability.

Stable atherosclerotic plaques are characterized by a thick, extracellular matrix-rich fibrous cap populated by protective ACTA2+ myofibroblast (MF)-like cells, assumed to be almost exclusively derived from smooth muscle cells (SMCs). Herein, we show that in murine and human lesions, 20% to 40% of ACTA2+ fibrous cap cells, respectively, are derived from non-SMC sources, including endothelial cells (ECs) or macrophages that have undergone an endothelial-to-mesenchymal transition (EndoMT) or a macrophage-to-mesenchymal transition (MMT). In addition, we show that SMC-specific knockout of the Pdgfrb gene, which encodes platelet-derived growth factor receptor beta (PDGFRβ), in Apoe-/- mice fed a Western diet for 18 weeks resulted in brachiocephalic artery lesions nearly devoid of SMCs but with no changes in lesion size, remodelling or indices of stability, including the percentage of ACTA2+ fibrous cap cells. However, prolonged Western diet 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-sequencing analyses of the brachiocephalic artery region and in vitro models, we provide evidence that SMC-to-MF transitions are induced by PDGF and transforming growth factor-β and dependent on aerobic glycolysis, while EndoMT is induced by interleukin-1β and transforming growth factor-β. Together, we provide evidence that the ACTA2+ fibrous cap originates from a tapestry of cell types, which transition to an MF-like state through distinct signalling pathways that are either dependent on or associated with extensive metabolic reprogramming.

Objective:Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise toKlf4-dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO).Approach and Results:UsingMyh11-CreERT2eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP+cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP+; and unintended proinflammatory consequences. Using newly generatedMyh11-DreERT2tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specificKlf4knockout (smooth muscle cells and pericytesKlf4Δ/Δ) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1+lymphatic endothelial cells in the epididymal AT.Conclusions:Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.

Smooth muscle cells (SMC) are conventionally thought to promote atherosclerotic plaque stability by investing within the fibrous cap and protecting the blood flow from the harmful inflammatory plaque core. However, lineage tracing studies have shown that SMC in plaque can undergo a Klf4-dependent transition in vivo to a macrophage-like state, characterized by loss of SMC markers and expression of multiple macrophage markers, lipid accumulation, and phagocytic activity. Of major interest, SMC induced to the macrophage state in vitro by cholesterol loading also show increased production of the atheropromoting cytokine MCP1 (Monocyte chemoattractant protein 1). These results are surprising since it has been largely assumed that monocyte derived macrophages are the major source of MCP1 and that this acts to exacerbate lesion pathogenesis by inducing recruitment of additional circulating monocytes. Using MCP1-mCherry reporter mice on an ApoE -/- background, we show that MCP1 is highly expressed by medial and fibrous cap SMC. Further, we show that SMC-specific KLF4 knockout mice, which show marked reductions in the frequency of SMC-derived macrophages, show a 50% decrease in MCP1 immunostaining in late stage atherosclerotic lesions, suggesting that these cells are a major source of MCP-1 and produce it via a Klf4-dependent mechanism. Consistent with this possibility, we demonstrated that SMC-specific tamoxifen conditional MCP1 knockout mice on an ApoE -/- background (Myh11-CreERT2 eYFP ApoE -/- MCP1-mCherry), showed greatly decreased total MCP1 protein level within the aorta. We are currently completing examination of the effects of SMC specific MCP1 KO on late stage lesion pathogenesis in our Myh11-CreERT2 eYFP ApoE -/- MCP1-mCherry mice following 18 weeks of Western diet feeding. Taken together results provide evidence that SMC derived cells are an unexpected major source of MCP1 within lesions.