BACKGROUND:Phenotypic plasticity of smooth muscle cells (SMCs) and endothelial cells (ECs) contributes to atherosclerotic plaque composition and stability, yet how shifts in one population influence the contribution and function of the other under conditions of vascular stress, such as irradiation, is poorly understood. A major limitation has been the inability to simultaneously fate-map both cell types within the same lesion, with most studies mapping one lineage while inferring the other using unreliable dynamically changing marker genes, risking false-positive and false-negative assignment. METHODS:We generated dual lineage tracing Apoe-deficient mice, enabling simultaneous fate mapping of SMCs and ECs. This model was used to extend prior findings from single lineage tracing models demonstrating irradiation-induced loss of SMC lesion investment and expansion of EC-derived cells. Dual lineage tracing mice were subjected to irradiation followed by Western diet feeding to induce atherosclerosis. Lineage tracing, immunostaining, and single-cell RNA sequencing analysis were used to define coordinated SMC and EC responses and identify changes relevant to plaque instability. RESULTS:Dual lineage tracing simultaneously labeled SMC- and EC-derived cells in healthy and atherosclerotic vessels. Irradiation induced divergent responses: SMC-derived cells failed to invest in lesions and upregulated stress-activated inflammatory genes, whereas EC-derived cells expanded and upregulated SMC-associated genes. However, EC-derived cells within lesions failed to induce extracellular matrix genes, and lesions from irradiated mice exhibited reduced collagen content and fewer ACTA2 (α-smooth muscle actin)+ cells within the fibrous cap, consistent with reduced plaque stability. CONCLUSIONS:Dual lineage tracing of SMCs and ECs demonstrated that irradiation-induced loss of lesional SMC and expansion of EC-derived ACTA2+ cells are not artifacts of false lineage assignment. By resolving SMC and EC fate within the same lesion, we identify irradiation-induced cell dynamics including inflammatory reprogramming of SMCs, EC phenotypic modulation, impaired extracellular matrix organization, and reduced ACTA2+ fibrous cap cellularity that may contribute to radiotherapy-associated increased atherosclerotic cardiovascular disease risk.

BACKGROUND:Imatinib, the first Abl-tyrosine kinase inhibitor (TKI), improved leukemia outcomes without cardiovascular side effects. Newer agents, including ponatinib, addressed imatinib resistance, improving cancer remission, but substantially increased arterial thrombotic events, including myocardial infarction (MI) and stroke. The mechanism behind ponatinib-induced thrombosis and the cardiovascular effect of asciminib, a newly approved Abl-TKI, remain unknown. METHODS:The effect of clinically relevant plasma concentrations of imatinib, ponatinib, and asciminib were compared with vehicle in vivo using SR-BI-mut/LDLR-knockout (KO) mice to assess spontaneous MI and stroke risk. The mechanism was interrogated in C57BL/6J mice, assessing leukocyte trafficking and thromboinflammation by intravital microscopy and flow cytometry, respectively, and in ApoE-KO mice, assessing plaque phenotype by flow cytometry and histology. In vitro effects on human umbilical vein endothelial cells (ECs) and human coronary artery ECs were determined by flow cytometry, PCR, and immunoblotting. The role of TNF (tumor necrosis factor) signaling was evaluated by pharmacological inhibition and small interfering RNA knockdown. RESULTS:In SR-BI-mut/LDLR-KO mice, ponatinib significantly accelerated death from MI and stroke compared with vehicle, imatinib, and asciminib. In human ECs, only ponatinib increased expression of TNF receptors (TNFRs) and adhesion molecules (P-selectin, ICAM1 [intercellular adhesion molecule 1], and VCAM1 [vascular cell adhesion molecule 1]). Ponatinib rapidly induced TNFR2 membrane trafficking and TNF signaling in human umbilical vein ECs. TNFR inhibition or TNFR2 knockdown prevented ponatinib induction of EC adhesion molecules. In vivo, ponatinib increased mesenteric vessel adhesion molecules, leukocyte rolling and adhesion to vessels, leukocyte and platelet activation, and platelet-leukocyte aggregates. In ApoE-KO mice, ponatinib increased plaque necrotic core and inflammation, consistent with a rupture-prone phenotype. Asciminib-treated mice developed none of these in vitro or in vivo toxicities. In C57BL/6J mice, TNFR inhibition blocked ponatinib-induced mesenteric adhesion molecule expression and leukocyte trafficking, but not platelet-leukocyte aggregation. TNFR blockade prevented ponatinib-induced plaque inflammation in ApoE-KO mice and MI and stroke in SR-BI-mut/LDLR-KO mice. CONCLUSIONS:Ponatinib, a potent anticancer therapy, activates ECs, platelets, and leukocytes, driving plaque inflammation and death from MI and stroke in mice, mirroring clinical cardiotoxicities in patients with cancer. Asciminib did not induce these effects, suggesting it might be a safer option for imatinib-resistant patients with cancer. Inhibition of TNFR-mediated endothelial activation is sufficient to prevent ponatinib-induced major adverse cardiovascular events.

Background Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions are the leading cause of death in the world. The most common and effective means to reduce these major adverse cardiovascular events (MACE), including myocardial infarction (MI) and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, little is known regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. Methods Smooth muscle cell (SMC) - lineage tracing Apoe −/− mice were fed a Western diet (WD) for 18 weeks and then switched to a low-fat chow diet for 12 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery (BCA) lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of SMC, and other lesion cells by SMC-lineage tracing combined with scRNA-seq, CyTOF, and immunostaining plus high resolution confocal microscopic z-stack analysis. In addition, to determine if treatment with a potent inhibitor of inflammation could augment the benefits of chow diet-induced reductions in LDL-cholesterol, SMC - lineage tracing Apoe −/− mice were fed a WD for 18 weeks and then chow diet for 12 weeks prior to treating them with an IL-1β or control antibody (Ab) for 8-weeks. Results Lipid-lowering by switching Apoe −/− mice from a WD to a chow diet reduced LDL-cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1β Ab treatment resulted in multiple detrimental changes including increased plaque burden, BCA lesion size, as well as increased cholesterol crystal accumulation, intra-plaque hemorrhage, necrotic core area, and senescence as compared to IgG control Ab treated mice. Furthermore, IL-1β Ab treatment upregulated neutrophil degranulation pathways but down-regulated SMC extracellular matrix pathways likely important for the protective fibrous cap. Conclusions Taken together, IL-1β appears to be required for chow diet-induced reductions in plaque burden and increases in multiple indices of plaque stability. Clinical Perspective Although one must be cautious in extrapolating results of mouse studies to humans, the current and our previous studies (Gomez et al. 2018 Nature Medicine ) suggest that efforts to identify anti-inflammatory therapies for treating patients with advanced atherosclerosis should consider the possibility that inhibiting a given cytokine may have a mixture of beneficial and detrimental effects that vary between individuals. What is New? In a mouse model of advanced atherosclerosis followed by diet-induced reductions in cholesterol, IL-1β inhibition unexpectedly had multiple detrimental effects including increasing overall plaque burden and decreasing various indices of plaque stability, as well as markedly increasing the number of senescent cells, and cholesterol crystal accumulation in lesions as compared to IgG control Ab treated mice. What are the clinical Implications? Results suggest that some anti-inflammatory therapies may have limited efficacy for treating patients with advanced atherosclerosis because such therapies inhibit not only detrimental pro-inflammatory responses, but also evolutionarily conserved beneficial inflammatory processes, which play a critical role in resistance to pathogenic microorganisms, in tissue repair following injury, and resolution of inflammation. We propose that the latter includes clearance of senescent cells and cholesterol from advanced atherosclerotic lesions induced by dietary-induced lipid lowering.

BACKGROUND: Thromboembolic events, including myocardial infarction (MI) or stroke, caused by the rupture or erosion of unstable atherosclerotic plaques are the leading cause of death worldwide. Although most mouse models of atherosclerosis develop lesions in the aorta and carotid arteries, they do not develop advanced coronary artery lesions. Moreover, they do not undergo spontaneous plaque rupture with MI and stroke or do so at such a low frequency that they are not viable experimental models to study late-stage thrombotic events or to identify novel therapeutic approaches for treating atherosclerotic disease. This has stymied the development of more effective therapeutic approaches for reducing these events beyond what has been achieved with aggressive lipid lowering. Here, we describe a diet-inducible mouse model that develops widespread advanced atherosclerosis in coronary, brachiocephalic, and carotid arteries with plaque rupture, MI, and stroke. METHODS: We characterized a novel mouse model with a C-terminal mutation in the scavenger receptor class B, type 1 (SR-BI), combined with Ldlr knockout (designated SR-BI ∆CT/∆CT / Ldlr −/− ). Mice were fed Western diet (WD) for 26 weeks and analyzed for MI and stroke. Coronary, brachiocephalic, and carotid arteries were analyzed for atherosclerotic lesions and indices of plaque stability. To validate the utility of this model, SR-BI ∆CT/∆CT / Ldlr −/− mice were treated with the drug candidate AZM198, which inhibits myeloperoxidase, an enzyme produced by activated neutrophils that predicts rupture of human atherosclerotic lesions. RESULTS: SR-BI ∆CT/∆CT / Ldlr −/− mice show high (>80%) mortality rates after 26 weeks of WD feeding because of major adverse cardiovascular events, including spontaneous plaque rupture with MI and stroke. Moreover, WD-fed SR-BI ∆CT/∆CT / Ldlr −/− mice displayed elevated circulating high-sensitivity cardiac troponin I and increased neutrophil extracellular trap formation within lesions compared with control mice. Treatment of WD-fed SR-BI ∆CT/∆CT / Ldlr −/− mice with AZM198 showed remarkable benefits, including >90% improvement in survival and >60% decrease in the incidence of plaque rupture, MI, and stroke, in conjunction with decreased circulating high-sensitivity cardiac troponin I and reduced neutrophil extracellular trap formation within lesions. CONCLUSIONS: WD-fed SR-BI ∆CT/∆CT / Ldlr −/− mice more closely replicate late-stage clinical events of advanced human atherosclerotic disease than previous models and can be used to identify and test potential new therapeutic agents to prevent major adverse cardiac events.

Domain adaptive semantic segmentation aims to generate accurate and dense predictions for an unlabeled target domain by leveraging a supervised model trained on a labeled source domain. The prevalent self-training approach involves retraining the dense discriminative classifier of p(class|pixel feature) using the pseudo-labels from the target domain. While many methods focus on mitigating the issue of noisy pseudo-labels, they often overlook the underlying data distribution p(pixel feature|class) in both the source and target domains. To address this limitation, we propose the multi-prototype Gaussian-Mixture-based (ProtoGMM) model, which incorporates the GMM into contrastive losses to perform guided contrastive learning. Contrastive losses are commonly executed in the literature using memory banks, which can lead to class biases due to underrepresented classes. Furthermore, memory banks often have fixed capacities, potentially restricting the model's ability to capture diverse representations of the target/source domains. An alternative approach is to use global class prototypes (i.e. averaged features per category). However, the global prototypes are based on the unimodal distribution assumption per class, disregarding within-class variation. To address these challenges, we propose the ProtoGMM model. This novel approach involves estimating the underlying multi-prototype source distribution by utilizing the GMM on the feature space of the source samples. The components of the GMM model act as representative prototypes. To achieve increased intra-class semantic similarity, decreased inter-class similarity, and domain alignment between the source and target domains, we employ multi-prototype contrastive learning between source distribution and target samples. The experiments show the effectiveness of our method on UDA benchmarks.

Aging is known to exacerbate atherosclerosis, but the mechanisms have been largely unknown. A study in Nature Aging reveals a bone-marrow-controlled axis of clonality during atherosclerosis, showing that aged bones drive an inflammatory milieu that promotes smooth muscle polyclonality and the formation of larger lesions.

Recently, deep learning-based methods achieved promising performance in nuclei detection and classification applications. However, training deep learning-based methods requires a large amount of pixel-wise annotated data, which is time-consuming and labor-intensive, especially in 3D images. An alternative approach is to adapt weak-annotation methods, such as labeling each nucleus with a point, but this method does not extend from 2D histopathology images (for which it was originally developed) to 3D immunofluorescent images. The reason is that 3D images contain multiple channels (z-axis) for nuclei and different markers separately, which makes training using point annotations difficult. To address this challenge, we propose the Label-efficient Contrastive learning-based (LECL) model to detect and classify various types of nuclei in 3D immunofluorescent images. Previous methods use Maximum Intensity Projection (MIP) to convert immunofluorescent images with multiple slices to 2D images, which can cause signals from different z-stacks to falsely appear associated with each other. To overcome this, we devised an Extended Maximum Intensity Projection (EMIP) approach that addresses issues using MIP. Furthermore, we performed a Supervised Contrastive Learning (SCL) approach for weakly supervised settings. We conducted experiments on cardiovascular datasets and found that our proposed framework is effective and efficient in detecting and classifying various types of nuclei in 3D immunofluorescent images.

Thromboembolic events, including myocardial infarction (MI) or stroke, caused by the rupture or erosion of unstable atherosclerotic plaques are the leading cause of death worldwide 1 . Unfortunately, the lack of a mouse model that develops advanced coronary atherosclerosis and that exhibits a high incidence of spontaneous plaque rupture with MI or stroke has greatly stymied development of more effective therapeutic approaches for reducing these events beyond what has been achieved with aggressive lipid lowering. Herein, we describe a novel mouse model that develops widespread advanced atherosclerosis including in coronary, brachiocephalic, and carotid arteries. These mice show high mortality following Western Diet feeding with clear evidence of plaque rupture, MI, and stroke. To validate the utility of this model, mice were treated with the drug candidate AZM198, which inhibits myeloperoxidase, an enzyme primarily produced by activated neutrophils and predictive of rupture of human atherosclerotic lesions 2–7 . AZM198 treatment resulted in marked improvements in survival with a greater than 60% decrease in the incidence of plaque rupture, MI, and stroke. In summary, our work describes a novel mouse model that closely replicates late-stage clinical events of advanced human atherosclerotic disease and evidence that this model can be used to identify and test potential new therapeutic agents to prevent major adverse cardiac events.

Background: Smooth muscle cells (SMCs) in atherosclerotic plaque take on multiple nonclassical phenotypes that may affect plaque stability and, therefore, the likelihood of myocardial infarction or stroke. However, the mechanisms by which these cells affect stability are only beginning to be explored. Methods: In this study, we investigated the contribution of inflammatory MCP1 (monocyte chemoattractant protein 1) produced by both classical Myh11 (myosin heavy chain 11)+ SMCs and SMCs that have transitioned through an Lgals3 (galectin 3)+ state in atherosclerosis using smooth muscle lineage tracing mice that label all Myh11+ cells and a dual lineage tracing system that targets Lgals3-transitioned SMC only. Results: We show that loss of MCP1 in all Myh11+ smooth muscle results in a paradoxical increase in plaque size and macrophage content, driven by a baseline systemic monocytosis early in atherosclerosis pathogenesis. In contrast, knockout of MCP1 in Lgals3-transitioned SMCs using a complex dual lineage tracing system resulted in lesions with an increased Acta2 (actin alpha 2, smooth muscle)+ fibrous cap and decreased investment of Lgals3-transitioned SMCs, consistent with increased plaque stability. Finally, using flow cytometry and single-cell RNA sequencing, we show that MCP1 produced by Lgals3-transitioned SMCs influences multiple populations of inflammatory cells in late-stage plaques. Conclusions: MCP1 produced by classical SMCs influences monocyte levels beginning early in disease and was atheroprotective, while MCP1 produced by the Lgals3-transitioned subset of SMCs exacerbated plaque pathogenesis in late-stage disease. Results are the first to determine the function of Lgals3-transitioned inflammatory SMCs in atherosclerosis and highlight the need for caution when considering therapeutic interventions involving MCP1.

The fat mass and obesity gene (FTO) is a N-6-methyladenosine RNA demethylase that was initially linked by Genome-wide association studies to increased rates of obesity. Subsequent studies have revealed multiple mass-independent effects of the gene, including cardiac myocyte contractility. We created a mouse with a conditional and inducible smooth muscle cell deletion of Fto (Myh11 Cre(+) Fto(fl/fl)) and did not observe any changes in mouse body mass or mitochondrial metabolism. However, the mice had significantly decreased blood pressure (hypotensive), despite increased heart rate and sodium, and significantly increased plasma renin. Remarkably, the third-order mesenteric arteries from these mice had almost no myogenic tone or capacity to constrict to smooth muscle depolarization or phenylephrine. Microarray analysis from Fto(-/-)-isolated smooth muscle cells demonstrated a significant decrease in serum response factor (Srf) and the downstream effectors Acta2, Myocd, and Tagln; this was confirmed in cultured human coronary arteries with FTO siRNA. We conclude Fto is an important component to the contractility of smooth muscle cells. NEW & NOTEWORTHY We show a key role for the fat mass obesity (FTO) gene in regulating smooth muscle contractility, possibly by methylation of serum response factor (Srf).

HomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 43, No. 1Response by Owens and Deaton to Letter Regarding Article, "Dichotomous Roles of Smooth Muscle Cell–Derived MCP1 (Monocyte Chemoattractant Protein 1) in Development of Atherosclerosis" Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBResponse by Owens and Deaton to Letter Regarding Article, "Dichotomous Roles of Smooth Muscle Cell–Derived MCP1 (Monocyte Chemoattractant Protein 1) in Development of Atherosclerosis" Gary K. Owens and Rebecca A. Deaton Gary K. OwensGary K. Owens https://orcid.org/0000-0002-7119-9657 University of Virginia School of Medicine, Robert M. Berne Cardiovascular Research Center, Charlottesville, VA. and Rebecca A. DeatonRebecca A. Deaton https://orcid.org/0000-0001-7569-1924 University of Virginia School of Medicine, Robert M. Berne Cardiovascular Research Center, Charlottesville, VA. Originally published21 Dec 2022https://doi.org/10.1161/ATVBAHA.122.318638Arteriosclerosis, Thrombosis, and Vascular Biology. 2023;43:e64In Response:We thank Wang et al for their kind remarks and further insights regarding our Owsiany et al1 2022 Arteriosclerosis, Thrombosis, and Vascular Biology article. We fully agree with virtually all their comments including there being multiple possible cellular sources of MCP1 (monocyte chemoattractant protein 1) and secondary responses to its knockout in smooth muscle cells (SMC) including activating other cytokines. As is the case with any gene knockout study, the phenotype observed is a function not only of the initial gene knockout but also any downstream adaptive and maladaptive responses. Our observation that SMC heterozygous, but not homozygous MCP1 knockout mice, showed a paradoxical increase in plaque size and macrophage content is consistent with this idea since the homozygous SMC MCP1 knockout mice are more likely to undergo compensatory changes. Moreover, these results unveiled an unexpected beneficial role of SMC-derived MCPI in a Western diet–fed Apoe−/− mouse model. We present evidence, but certainly not proof, that this phenotype may be the result of systemic monocytosis secondary to loss of SMC-derived MCP1 causing increased release of monocytes from hematopoietic stem cell niches. However, as indicated by Wang et al, there are numerous other possibilities that remain to be tested. One interesting possibility is that initial production of MCP1 by SMC is beneficial because it promotes recruitment of monocytes-macrophages to early-stage atherosclerotic lesions as a means to remove oxidized lipids and apoptotic cells. However, as suggested by Randolph,2 the process may only be effective for a short time before the macrophages are overwhelmed, become engorged with lipids, fail to egress to the lymphatics, and give rise to foam cells and contribute to a chronic inflammatory state.We also agree with their comment that there is stage-specific regulation due to the different cellular sources of MCP1 at different stages of atherosclerosis. In support of this idea, we observed that mice with MCP1 knockout in the subset of SMC that have transitioned through a Lgals3 (Galectin3)+ state exhibit a phenotype virtually opposite to that of mice with MCP1 knockout in all SMC. SMC-Lgals3 dual recombinase MCP1 knockout mice had lesions with an increased ACTA2 (smooth muscle alpha-actin)+ 2 fibrous cap and decreased investment of Lgals3-transitioned SMCs, consistent with increased plaque stability. That is, MCP1 expression by this subset of SMC appears to be detrimental.Finally, we wish to caution readers to be careful in directly comparing results of cell-specific conditional gene knockout to global conventional gene knockout studies given the many undefined variables between such models including the timing of gene knockout and complex differences in the phenotypic state of lesion cells at any given time point. Our SMC-specific conditional MCP1 knockout mouse models provide valuable mechanistic insights regarding the contributions of SMC-derived MCP1 to lesion pathogenesis. However, these models are not intended or likely to predict overall effects of systemic MCP1 inhibitors.Article InformationDisclosures None.References1. Owsiany KM, Deaton RA, Soohoo KG, Tram NA, Owens GK. Dichotomous roles of smooth muscle cell-derived MCP1 (monocyte chemoattractant protein 1) in development of atherosclerosis.Arterioscler Thromb Vasc Biol. 2022; 42:942–956. doi: 10.1161/ATVBAHA.122.317882LinkGoogle Scholar2. Randolph GJ. Emigration of monocyte-derived cells to lymph nodes during resolution of inflammation and its failure in atherosclerosis.Curr.Opin.Lipidol. 2008 Oct; 19(5):462–468. doi: 10.1097/MOL.0b013e32830d5f09CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails January 2023Vol 43, Issue 1 Advertisement Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/ATVBAHA.122.318638PMID: 36542726 Originally publishedDecember 21, 2022 PDF download Advertisement

Background: The Myh11 promoter is extensively used as a smooth muscle cell (SMC) Cre-driver and is regarded as the most restrictive and specific promoter available to study SMCs. Unfortunately, in the existing Myh11-CreER T2 mouse, the transgene was inserted on the Y chromosome precluding the study of female mice. Given the importance of including sex as a biological variable and that numerous SMC-based diseases have a sex-dependent bias, the field has been tremendously limited by the lack of a model to study both sexes. Here, we describe a new autosomal Myh11-CreER T2 mouse (referred to as Myh11-CreER T2 -RAD ), which allows for SMC-specific lineage tracing and gene knockout studies in vivo using both male and female mice. Methods: A Myh11-CreER T2 -RAD transgenic C57BL/6 mouse line was generated using bacterial artificial chromosome clone RP23-151J22 modified to contain a Cre-ER T2 after the Myh11 start codon. Myh11-CreER T2 -RAD mice were crossed with 2 different fluorescent reporter mice and tested for SMC-specific labeling by flow cytometric and immunofluorescence analyses. Results: Myh11-CreER T2 -RAD transgene insertion was determined to be on mouse chromosome 2. Myh11-CreER T2 -RAD fluorescent reporter mice showed Cre-dependent, tamoxifen-inducible labeling of SMCs equivalent to the widely used Myh11-CreER T2 mice. Labeling was equivalent in both male and female Cre + mice and was limited to vascular and visceral SMCs and pericytes in various tissues as assessed by immunofluorescence. Conclusions: We generated and validated the function of an autosomal Myh11-CreER T2 -RAD mouse that can be used to assess sex as a biological variable with respect to the normal and pathophysiological functions of SMCs.

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. Newman et al. explore the origins of myofibroblasts in atherosclerotic fibrous caps, finding that while composed of cells from multiple origins, smooth muscle cells predominate and are required for long-term plaque stability.

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.

Objective: Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4 -dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and Results: Using Myh11-Cre ERT2 eYFP (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 generated Myh11-Dre ERT2 tdTomato 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-specific Klf4 knockout (smooth muscle cells and pericytes Klf4 Δ/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.

There is clear evidence that the phenotypic modulation of smooth muscle cells (SMCs) contributes to the pathophysiology of vascular disease. Phenotypic modulation refers to the unique ability of SMCs to alter their phenotype in response to extracellular stimuli and is hallmarked by the loss of SMC marker gene expression. The transcription factor Krüppel-like factor 4 (KLF4) is a known powerful negative regulator of SMC marker gene expression that works, in part, by decreasing the expression of the serum response factor (SRF) myocardin. KLF4 is not expressed in healthy adult SMCs but is increased in SMCs in response to vascular injury in vivo or PDGF-BB treatment in vitro. The aim of the present study was to determine the molecular mechanisms that regulate the expression of KLF4 in phenotypically modulated SMCs. The results demonstrated that the transcription factor stimulating protein-1 (Sp1) regulated the expression of KLF4 in SMCs. The KLF4 promoter contains three consensus Sp1 binding sites. Using a series of truncated KLF4 promoters, we showed that only fragments containing these Sp1 sites could be activated by PDGF-BB. In addition, overexpression of Sp1 alone was sufficient to increase the activity of the KLF4 promoter. Moreover, inhibiting Sp1 expression with small-interfering RNA attenuated the effects of PDGF-BB on KLF4 expression. Mutation of the three Sp1 sites within the KLF4 promoter abolished both baseline and PDGF-BB-induced activity. Finally, the results demonstrated enhanced Sp1 binding to the KLF4 promoter in SMCs treated with PDGF-BB in vitro and following vascular injury in vivo. Taken together, the results suggest a novel role for Sp1 in increasing the expression of KLF4 in phenotypically modulated SMCs.