SMC-derived Cells Research Articles

Smooth muscle cells clonally expand in a murine carotid allograft model complicated by immune reactions to reporter transgenes

Background and aimsMost experimental studies of allograft vasculopathy (AV) have relied on transplantation between major histocompatibility complex-mismatched inbred mouse strains, but this leads to the complete eradication of donor smooth muscle cells (SMCs) and lesions formed by recipient cells. This is unlike human AV which is thought to form mainly by donor SMCs. Here, we studied sources of neointimal cells in a minor histocompatibility antigen-mismatched AV model by combining male-to-female orthotopic carotid transplantations and lineage tracing by SMC-specific expression of fluorescent proteins. MethodsTo track SMC-derived cells in allograft vasculopathy, we used male donor mice with SMC-restricted Cre recombination of the mT/mG reporter transgene, which switches expression of membrane-bound red fluorescent protein (RFP) to green fluorescent protein (GFP), or the stochastically recombining Confetti reporter transgene, which yields a mosaic expression of four fluorescent proteins. Donor carotid segments were harvested and orthotopically allografted to female recipients that were wildtype or had non-recombined reporter transgenes. Inhibition of T cell responses by CTLA4Ig was used in some experiments. Sections of lesions harvested after 4 weeks were analyzed by fluorescence microscopy. ResultsDonor-derived SMCs survived and gave rise to part of the neointimal cells in experiments where carotid segments from recombined mT/mG male mice were transplanted into wild-type or non-recombined mT/mG female mice. Sex-mismatched transplants developed significant lesions, increasing the intimal and medial area 4.6-fold (p = 0.038) and 2.0-fold (p = 0.024) compared to sex- and fluorescence-matched controls, respectively. Interestingly, sex-matched fluorescence-positive transplants developed intimal lesions in 50% of fluorescence-naïve recipient controls. To study the clonal structure of the neointimal donor-derived SMC lineage cells, we then transplanted male carotids with heterozygous or homozygous recombined Confetti transgenes into female recipients. These transplants developed lesions with few surviving donor SMCs, indicating that expression of the Confetti reporter increased rejection and donor-specific SMC death. Some of the few remaining donor SMCs underwent clonal expansion. CTLA4Ig administration at the time of surgery did not improve SMC survival in mT/mG or Confetti transplants. ConclusionMale-to-female transplant models feature donor-derived SMCs, some of which undergo clonal expansion, but immune rejection to fluorescence reporters appears to bias results in lineage tracing models. Overcoming these challenges with alternative reporter transgenes or tolerant recipients is necessary to study the mechanisms by which donor SMCs contribute to allograft vasculopathy.

Abstract 1043: Single-cell Characterization Of Mouse Aorta During Atherosclerosis Regression

Atherosclerosis is a cardiovascular disease characterized by the accumulation of fibro-fatty plaque in the arteries. Over half the cells in a mature atherosclerotic lesion are smooth muscle cells (SMCs) or SMC-derived cells (SDCs). SMCs can undergo a process called phenotypic switching, which involves dedifferentiation to an intermediate multipotent cell state, migration, and transdifferentiation into alternative cell types (SDCs). Increased SMC phenotypic switching is associated with worse atherosclerosis progression. However, little is known of the roles of SMCs and SDCs in atherosclerosis regression. We hypothesize that SDCs may promote plaque stability during atherosclerosis regression. In this study, we used an SMC lineage tracing mouse model (Ldlr–/–; LoxP-stop-LoxP-ZsGreen1+/-; Myh11-CreERT2+/-) that is limited to male mice. At 6 weeks of age, the mice were administered a tamoxifen diet for 1 week, followed by a week of chow diet. Mice were then fed a 16-week Western diet to induce atherosclerosis. We modeled lipid-lowering induced regression by switching to chow diet and administering HDAd-hLDLR. Mice were harvested at timepoints 0, 2, 5, 10, and 20 weeks. Arterial tissues, such as the aorta, brachiocephalic artery, and aortic root were used for histological characterization of disease and for single cell RNA-sequencing (scRNA-seq). The scRNA-seq data showed no major changes in SMC and SDC cell clusters, showing that our hypothesis was likely null. We noted an overall decrease in macrophages, and an increase in both of the two fibroblast clusters, which also had transcriptional signatures of increased proliferation and reduced apoptosis and senescence. Our histology data showed thicker fibrous caps and increased collagen, as expected in a regression model. Going forward, we will assess whether one or both fibroblast clusters are necessary for the stabilization of plaques during atherosclerosis regression.

Abstract 157: Atherosclerosis Is A Smooth Muscle Cell-driven Tumor-like Disease

Background: Atherosclerosis, a leading cause of cardiovascular disease, involves the pathological activation of various cell types, including immunocytes (e.g., macrophages, T cells), smooth muscle cells (SMCs), and endothelial cells. Accumulating evidence suggests that transition of SMCs to other cell types, known as “phenotypic switching,” plays a central role in atherosclerosis development and complications. However, the characteristics of SMC-derived cells and the underlying mechanisms of SMC transition in disease pathogenesis remain poorly understood. Our objective is to characterize tumor cell-like behaviors of SMC-derived cells in atherosclerosis, with the ultimate goal of developing interventions targeting SMC transition for the prevention and treatment of atherosclerosis. Methods: We used SMC lineage tracing mice and human tissues and applied a range of methods, including molecular, cellular, histological, computational, human genetics, and pharmacological approaches to investigate the features of SMC-derived cells in atherosclerosis. Results: SMC-derived cells in mouse and human atherosclerosis exhibit multiple tumor cell-like characteristics, including genomic instability, evasion of senescence, hyperproliferation, resistance to cell death, invasiveness, and activation of comprehensive cancer-associated gene regulatory networks. Specific expression of oncogenic mutant, Kras G12D , in SMCs accelerates phenotypic switching and exacerbates atherosclerosis. Furthermore, we provide a proof of concept that niraparib, an anti-cancer drug targeting DNA damage repair, attenuates atherosclerosis progression and induces regression of lesions in advanced disease in mouse models. Conclusions: Our findings demonstrate that atherosclerosis is a SMC-driven tumor-like disease, advancing our understanding of its pathogenesis and opening prospects for innovative precision molecular strategies aimed at preventing and treating atherosclerotic cardiovascular disease.

Atherosclerosis Is a Smooth Muscle Cell-Driven Tumor-Like Disease.

Atherosclerosis, a leading cause of cardiovascular disease, involves the pathological activation of various cell types, including immunocytes (eg, macrophages and T cells), smooth muscle cells (SMCs), and endothelial cells. Accumulating evidence suggests that transition of SMCs to other cell types, known as phenotypic switching, plays a central role in atherosclerosis development and complications. However, the characteristics of SMC-derived cells and the underlying mechanisms of SMC transition in disease pathogenesis remain poorly understood. Our objective is to characterize tumor cell-like behaviors of SMC-derived cells in atherosclerosis, with the ultimate goal of developing interventions targeting SMC transition for the prevention and treatment of atherosclerosis. We used SMC lineage tracing mice and human tissues and applied a range of methods, including molecular, cellular, histological, computational, human genetics, and pharmacological approaches, to investigate the features of SMC-derived cells in atherosclerosis. SMC-derived cells in mouse and human atherosclerosis exhibit multiple tumor cell-like characteristics, including genomic instability, evasion of senescence, hyperproliferation, resistance to cell death, invasiveness, and activation of comprehensive cancer-associated gene regulatory networks. Specific expression of the oncogenic mutant KrasG12D in SMCs accelerates phenotypic switching and exacerbates atherosclerosis. Furthermore, we provide proof of concept that niraparib, an anticancer drug targeting DNA damage repair, attenuates atherosclerosis progression and induces regression of lesions in advanced disease in mouse models. Our findings demonstrate that atherosclerosis is an SMC-driven tumor-like disease, advancing our understanding of its pathogenesis and opening prospects for innovative precision molecular strategies aimed at preventing and treating atherosclerotic cardiovascular disease.

High-Dimensional Single-Cell Multimodal Landscape of Human Carotid Atherosclerosis.

Atherosclerotic plaques are complex tissues composed of a heterogeneous mixture of cells. However, our understanding of the comprehensive transcriptional and phenotypic landscape of the cells within these lesions is limited. To characterize the landscape of human carotid atherosclerosis in greater detail, we combined cellular indexing of transcriptomes and epitopes by sequencing and single-cell RNA sequencing to classify all cell types within lesions (n=21; 13 symptomatic) to achieve a comprehensive multimodal understanding of the cellular identities of atherosclerosis and their association with clinical pathophysiology. We identified 25 cell populations, each with a unique multiomic signature, including macrophages, T cells, NK (natural killer) cells, mast cells, B cells, plasma cells, neutrophils, dendritic cells, endothelial cells, fibroblasts, and smooth muscle cells (SMCs). Among the macrophages, we identified 2 proinflammatory subsets enriched in IL-1B (interleukin-1B) or C1Q expression, 2 TREM2-positive foam cells (1 expressing inflammatory genes), and subpopulations with a proliferative gene signature and SMC-specific gene signature with fibrotic pathways upregulated. Further characterization revealed various subsets of SMCs and fibroblasts, including SMC-derived foam cells. These foamy SMCs were localized in the deep intima of coronary atherosclerotic lesions. Utilizing cellular indexing of transcriptomes and epitopes by sequencing data, we developed a flow cytometry panel, using cell surface proteins CD29, CD142, and CD90, to isolate SMC-derived cells from lesions. Lastly, we observed reduced proportions of efferocytotic macrophages, classically activated endothelial cells, and contractile and modulated SMC-derived cells, while inflammatory SMCs were enriched in plaques of clinically symptomatic versus asymptomatic patients. Our multimodal atlas of cell populations within atherosclerosis provides novel insights into the diversity, phenotype, location, isolation, and clinical relevance of the unique cellular composition of human carotid atherosclerosis. These findings facilitate both the mapping of cardiovascular disease susceptibility loci to specific cell types and the identification of novel molecular and cellular therapeutic targets for the treatment of the disease.

Cholesterol lowering depletes atherosclerotic lesions of smooth muscle cell-derived fibromyocytes and chondromyocytes.

Drugs that lower plasma apolipoprotein B (ApoB)-containing lipoproteins are central to treating advanced atherosclerosis and provide partial protection against clinical events. Previous research showed that lowering ApoB-containing lipoproteins stops plaque inflammation, but how these drugs affect the heterogeneous population of plaque cells derived from smooth muscle cells (SMCs) is unknown. SMC-derived cells are the main cellular component of atherosclerotic lesions and the source of structural components that determine the size of plaques and their propensity to rupture and trigger thrombosis, the proximate cause of heart attack and stroke. Using lineage tracing and single-cell techniques to investigate the full SMC-derived cellular compartment in progressing and regressing plaques in mice, here we show that lowering ApoB-containing lipoproteins reduces nuclear factor kappa-light-chain-enhancer of activated B cells signaling in SMC-derived fibromyocytes and chondromyocytes and leads to depletion of these abundant cell types from plaques. These results uncover an important mechanism through which cholesterol-lowering drugs can achieve plaque regression.

Abstract 701: Atherosclerosis Is A Smooth Muscle Cell-driven Tumor-like Disease

Background: Atherosclerosis, the leading cause of cardiovascular disease (CVD), is a chronic inflammatory disease involving pathological activation of multiple cell types, such as immunocytes (e.g., macrophage, T cells), smooth muscle cells (SMCs), endothelial cells, etc. Multiple lines of evidence have suggested that SMC “phenotypic switching” plays a central role in atherosclerosis development and complications. Yet, how SMCs and SMC-derived cells (SDCs) modulate atherosclerosis progression and clinical CVD remains poorly understood. Methods: We applied SMC lineage tracing mice and implemented comprehensive molecular and cellular biology, histological, computational, and pharmacological studies to reveal how tumor cell-like activities of SDCs drive the development of atherosclerosis. Results: Oxidative DNA damage occurs during SMC phenotypic switching and increases with the progression of atherosclerosis. Extensive DNA damage-induced genomic instability accumulates in both mouse and human atherosclerosis, especially in SDCs. Indeed, SDCs in atherosclerosis harbor multiple tumor cell-like features, including escape of replicative senescence, clonogenicity, invasive capacity, and ex vivo formation of three-dimensional (3D) spheroids like cancer stem cells. Multiple cancer-associated signaling pathways (e.g., p53, TNFa, NFkB, VEGF) are activated in SDCs. SMC-specific expression of oncogenic Kras G12D accelerates SMC phenotypic switching and exacerbates atherosclerosis. Moreover, we provide proof of concept in mouse models that niraparib, an anti-cancer drug targeting DNA damage repair, attenuates atherosclerosis progression and regresses lesions in advanced disease. Conclusions: Our studies in human and mouse models provide the first systematic evidence that SDCs in atherosclerosis share extensive commonalities with tumor, which deepens our understanding of the disease and opens prospects for novel targeted strategies to prevent and treat atherosclerotic CVD.

Abstract 720: Integration Of Population Genetic And Human Knockout Studies With Smooth Muscle Cell Lineage Tracing In Mouse Models Identifies Novel Targets For Atherosclerosis And Cardiovascular Risk.

Despite lipid-lowering therapies, residual cardiovascular disease risk remains a major unmet clinical need. Large-scale genetic analyses have identified >250 loci for coronary artery disease (CAD) risk. Genes that function in vascular smooth muscle cells (SMC) are causal at several loci, yet the causal genes at most loci remain unknown. Using single cell profiling and SMC lineage tracing in mouse models, we and others have shown that SMC transition through an intermediate state to SMC-derived cells (SDC) with protective or harmful phenotypes for plaque stability. In this study, we integrated SMC lineage tracing in mouse models with ongoing analyses in > 1 million human participants with whole-genome (WGS) and whole-exome sequencing (WES) data, as well as rare variant analysis of human “knockouts” (homozygous for predicted loss-of-function variants) for a novel gene discovery pipeline (Figure 1). Through WES in 76,000 highly consanguineous participants in the Pakistan Genomic Resource (PGR), we assembled the largest global cohort of human KOs. At non-lipid CAD loci from a recent large multi-ethnic meta-analysis (~1.1 million participants), we prioritized genes as potentially causal in SMC/SDC as those that are expressed and regulated in human or mouse arteries and SMC in atherosclerosis progression. Through gene burden testing and analyses of rare predicted loss-of-function (pLoF) and damaging (pDMs) variants in the largest CAD WES/WGS meta-analyses we enriched for causal genes. We restricted to genes with at least 5 pLoF carriers in PGR to facilitate recall studies in Pakistan. Final prioritization incorporated state-of-the-art fine mapping and co-localization analyses as well as biological plausibility. From an initial pool of 1696 genes, our gene discovery pipeline prioritized 15 SMC/SDC genes, and initial recall in PGR identified multiple large pedigrees for mutations in candidate causal genes PDE3A, SERPINH1, HHIPL1, and ZEB2.

Translating ribosome affinity purification identifies markers of atherosclerosis-associated smooth muscle cells

Background and Aims: Vascular smooth muscle cells (SMCs) and SMC-derived cells are a major source of plaque cells and extracellular matrix at all stages of atherosclerosis. Recent single cell sequencing studies have identified new subsets of SMCs and demonstrated disease-associated changes in gene expression. Still, single cell studies are limited by the high cost and tissue dissociation artefacts. To address these limitations, we developed a mouse model that allows cost-effective analysis of SMC-specific translatome from any tissue using Translating Ribosome Affinity Purification (TRAP-Seq).

Fibrous Caps in Atherosclerosis Form by Notch-Dependent Mechanisms Common to Arterial Media Development.

Atheromatous fibrous caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We tested whether the recruitment mechanisms are the same as in embryonic artery development, which relies prominently on Notch signaling to form the subendothelial medial SMC layers. Notch elements were expressed in regions of fibrous cap in human and mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice where the Notch pathway was inactivated in SMCs by conditional knockout of the essential effector transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). The recruitment of cap SMCs was significantly reduced without major effects on plaque size. Lineage tracing revealed the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. Conversely, to analyze whether the loss of Notch signaling is required for SMC-derived cells to accumulate in atherogenesis, we studied atherosclerosis in mice with constitutive activation of Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development. Sequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the cap forms as a neo-media that restores the connection between endothelium and subendothelial SMCs, transiently disrupted in early atherogenesis.

Single-Cell Genomics Reveals a Novel Cell State During Smooth Muscle Cell Phenotypic Switching and Potential Therapeutic Targets for Atherosclerosis in Mouse and Human.

Smooth muscle cells (SMCs) play significant roles in atherosclerosis via phenotypic switching, a pathological process in which SMC dedifferentiation, migration, and transdifferentiation into other cell types. Yet how SMCs contribute to the pathophysiology of atherosclerosis remains elusive. To reveal the trajectories of SMC transdifferentiation during atherosclerosis and to identify molecular targets for disease therapy, we combined SMC fate mapping and single-cell RNA sequencing of both mouse and human atherosclerotic plaques. We also performed cell biology experiments on isolated SMC-derived cells, conducted integrative human genomics, and used pharmacological studies targeting SMC-derived cells both in vivo and in vitro. We found that SMCs transitioned to an intermediate cell state during atherosclerosis, which was also found in human atherosclerotic plaques of carotid and coronary arteries. SMC-derived intermediate cells, termed "SEM" cells (stem cell, endothelial cell, monocyte), were multipotent and could differentiate into macrophage-like and fibrochondrocyte-like cells, as well as return toward the SMC phenotype. Retinoic acid (RA) signaling was identified as a regulator of SMC to SEM cell transition, and RA signaling was dysregulated in symptomatic human atherosclerosis. Human genomics revealed enrichment of genome-wide association study signals for coronary artery disease in RA signaling target gene loci and correlation between coronary artery disease risk alleles and repressed expression of these genes. Activation of RA signaling by all-trans RA, an anticancer drug for acute promyelocytic leukemia, blocked SMC transition to SEM cells, reduced atherosclerotic burden, and promoted fibrous cap stability. Integration of cell-specific fate mapping, single-cell genomics, and human genetics adds novel insights into the complexity of SMC biology and reveals regulatory pathways for therapeutic targeting of SMC transitions in atherosclerotic cardiovascular disease.

Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a 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 sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected 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 chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 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-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds 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-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives 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.

Mature Vascular Smooth Muscle Cells, but Not Endothelial Cells, Serve as the Major Cellular Source of Intimal Hyperplasia in Vein Grafts.

Neointima formation is a primary cause of intermediate to late vein graft (VG) failure. However, the precise source of neointima cells in VGs remains unclear. Approach and Results: Herein we clarify the relative contributions of mature vascular smooth muscle cells (SMCs) and endothelial cells (ECs) to neointima formation in a mouse model of VG remodeling via the genetic-inducible fate mapping approaches. Regardless of the magnitude of neointima formation, the recipient arterial and the donor venous SMCs contributed ≈55% of the neointima cells at the anastomotic regions, whereas only donor venous SMCs donated ≈68% of the neointima cells at the middle bodies. A small portion of the SMC-derived cells became non-SMC cells, most likely vascular stem cells, and constituted 2% to 11% of the cells in each major layer of VGs. In addition, the recipient arterial ECs were the major cellular source of re-endothelialization but did not contribute to neointima formation. The donor venous ECs donated ≈17% neointima cells in the VGs with mild neointima formation and conditional media from ECs after endothelial-to-mesenchymal transition suppressed vascular SMC dedifferentiation. The recipient arterial and donor venous mature SMCs dominate but contribute distinctly to intimal hyperplasia at the anastomosis and the middle body regions of VGs. The recipient arterial ECs are the major cellular source of re-endothelialization but do not donate neointima formation in VGs. Only the donor venous ECs undergo endothelial-to-mesenchymal transition. Endothelial-to-mesenchymal transition is marginal for generating neointima cells but is likely required for controlling the quality of VG remodeling.

Abstract 17184: Nearly One Third of GWAS CAD Are Putative SMC Klf4 or Oct4 Targets in Advanced Atherosclerotic Lesions

Atherosclerosis is a disease of chronic inflammation and the leading cause of morbidity and mortality worldwide. Despite decades of research, our understanding of the mechanisms regulating plaque stability remains poor. Until recently, smooth muscle cells (SMCs) were thought to play an athero-protective role in lesion pathogenesis. However, rigorous lineage tracing has shown a significant portion (>80%) of SMC-derived cells within an advanced atherosclerotic lesion did not express detectable SMC markers. Furthermore, SMC-specific conditional KO of Klf4 resulted in reduction of several indices of plaque instability and SMC-derived macrophage-like cells. In contrast, conditional KO of Oct4 in SMCs presented a reduction of SMC and increased of Lgals3+ cells within lesions, as well as increase of several indices of plaque instability. Taken together, SMCs can have a beneficial or detrimental effect in lesion pathogenesis. Hypothesis: Combinatorial analysis of Oct4/Klf4 ChIP-seq and RNA-seq of advanced atherosclerotic lesions from our SMC-Klf4 and SMC-Oct4 mice provide a unique opportunity to identify genes that play a critical role in regulating beneficial or detrimental changes in SMC phenotype. Results: SMC-Oct4 target genes are enriched for migration pathways, while SMC-Klf4 target genes are enriched for inflammation pathways. Further, atherosclerosis pathway, among others, presented significant opposite patterns (SMC-Oct4-KO up-regulation and SMC-Klf4-KO down-regulation), and presented strong significant gene anti-correlation, corroborating our phenotypic observations. Remarkably, we cross-reference our ChIP-seq datasets with CAD GWAS and found that 54 of the 161 human loci were either Klf4 and/or Oct4 SMC targets, highly suggesting that CAD GWAS genes might be affecting SMC function in lesion development/pathogenesis. To validate our results, we used the STARNET dataset, and we observed that both Oct4 and Klf4 are significantly up-regulated in aorta of CAD patients compared to controls. In addition, co-expression analysis shows that one of the Klf4 network is highly related to human GWAS CAD, including genes such as LDLR, APOE, PCSK9, TRIB1, and others. Surprisingly, more than 35% of genes in this network also are present in our SMC Klf4 ChIP-seq dataset. Conclusions: our results suggests that: late stage atherosclerotic lesions of mouse and humans seems to be more similar than previously thought; and nearly 1/3 of CAD GWAS loci appear to be affecting SMC function and are potentially Klf4/Oct4-dependent.

KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis.

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.

Abstract 1: Oct4 is Re-activated in Smooth Muscle Cells through KLF4/Hif1α and Hydroxymethylation Mechanisms and Plays a Critical Role in Atherogenesis

The pluripotency factor Oct4 is a protein that regulates embryonic stem cell (ESC) totipotency and is the only gene thought to be indispensable for production of induced pluripotent stem (iPS) cells. Although there is evidence that Oct4 may be expressed in non-ESC, this is controversial and as yet there is no direct evidence that Oct4 plays a functional role within somatic cells. We demonstrate that Oct4 is markedly induced in cells within atherosclerotic lesions from ApoE-/- mice fed a high-fat diet and within human coronary artery atherosclerotic lesions. We show that smooth muscle cell (SMC)-specific conditional knockout (KO) of Oct4 within Western diet-fed ApoE-/- mice resulted in increased atherosclerotic lesion size and other changes consistent with decreased plaque stability. Moreover, using a novel SMC lineage tracing mouse developed in our lab, we also show that lesions from SMC-specific conditional Oct4 KO mice contained far fewer de-differentiated (Acta2-) SMCs likely as the result of impaired SMC de-differentiation and migration from the media into the intima and not to altered proliferation or apoptosis. Further mechanistic studies using in vitro and in vivo Chromatin Immunoprecipitation assays, glycosylation-coupled methylation sensitive qPCR and hMeDIP show that activation of Oct4 within SMCs and within atherosclerotic lsions was associated with hydroxymethylation of the Oct4 promoter and was Klf4- and Hif1α-dependent. In addition, using in situ hybridization/proximity ligation (ISH-PLA) assay developed in our lab we showed hydroxymethylation of the Oct4 promoter within individual SMC-derived cells in atherosclerotic lesions of our SMC-lineage tracing ApoE-/- mice. These results are the first to show that Oct4 plays a direct functional role in regulating phenotypic transitions in somatic cells, and provide molecular mechanisms required for Oct4 re-activation.

Abstract 149: Pluripotency Factors Krüppel-Like Factor 4 and Octamer-binding Transcription Factor 4 Alter Indices of Plaque Stability in Long Term Western Diet Fed Mice by Regulating Smooth Muscle Cell Phenotypes

The bulk of life threatening thrombotic events have been associated with disruption of the fibrous cap, an atheroprotective layer of smooth muscle α-actin positive (ACTA2+) cells that form around the plaque, and the presence of a large foam cell-laden necrotic core within the plaque. Despite the overwhelming research demonstrating that ACTA2+ cells are beneficial for plaque stability, and cells positive for macrophage-markers are detrimental, there are major ambiguities regarding the origins of these cells, and their role in lesion stability. To clearly define the contribution of smooth muscle cells (SMCs) within atherosclerotic lesions, we generated SMC specific lineage tracing Apoe-/- mice containing a SM myosin heavy chain ( Myh11 ) tamoxifen-inducible cre-recombinase gene and a floxed STOP ROSA eYFP gene ( Myh11 YFP ApoE-/- mice) thus allowing activation of eYFP exclusively in fully differentiated SMCs before the onset of atherosclerosis and subsequent determination of the fate of these cells and their progeny irrespective of continued expression of MYH11 or other SMC marker genes. Remarkably, our results reveal that 86% of SMCs cannot be identified using traditional SMC markers, such as ACTA2, and 23% of presumed macrophages (LGALS3+ cells) are derived from SMC origins. The last finding was confirmed in human coronary atheromas using the ISH-PLA approach. SMC specific knockout (KO) of the pluripotency factor Klf4 in Myh11 YFP ApoE-/- mice did not alter the frequency of phenotypically modulated (ACTA2-eYFP+) SMCs within atherosclerotic lesions of mice fed a high fat diet for 18 weeks, however, decreased the number of ACTA2-eYFP+ SMCs that expressed LGALS3, and increased several indices of plaque stability, suggesting a detrimental role for KLF4 in SMCs within atherosclerotic lesions. Conversely, SMC specific Oct4 KO resulted in a dramatic reduction in the number of ACTA2-eYFP+ SMCs within the lesion with marked decreases in indices of plaque stability. In summary results show that the majority of SMC-derived cells within advanced atherosclerotic lesions cannot be identified using conventional SMC marker genes, and that phenotypic switching of SMC during atherogenesis is differentially regulated by the pluripotency factors KLF4 and OCT4.

Abstract 47: Development and Use of a Novel Single-Cell Epigenetic Lineage Tracing Method to Identify Smooth Muscle--Derived Macrophage-Like Cells Within Human Atherosclerotic Lesions

Aim: Smooth muscle cells (SMC) possess remarkable phenotypic plasticity that allows adaptation to changing environmental cues. Lack of definitive SMC lineage tracing studies and inability to identify phenotypically modulated SMCs within lesions due to loss of SMC marker gene expression raise major questions regarding the role of SMC in vivo in atherosclerosis progression. We hypothesize that a subset of cells within lesions that express macrophage markers are derived from SMC not monocytes and play a key role in determining plaque stability. Methods: We developed a novel lineage tracing based on detection of H3K4dime of the SM MHC gene, a SMC-specific epigenetic lineage marker we have previously shown is stable during phenotypic switching in vitro. Detection of H3K4dime of the SM MHC locus was done using a Proximity ligation assay (PLA) developed in our lab with an antibody targeting the biotinylated DNA probe for the SM MHC locus in conjunction with an anti-H3K4dime antibody. Use of secondary antibodies conjugated with oligonucleotides induces formation of circular DNA that serve as template for amplification, allowing visualization of co-localization of H3K4dime and the SM MHC gene (Duolink). Our new lineage tracing is suitable with human paraffin-embedded tissue sections (n=4) allowing investigation of SMC fate within human atherosclerotic lesion. Results: H3K4dime on the SM MHC gene (PLA+ cells) was found to be specific for SMCs and not found in any other cell types including adventitial fibroblasts, or endothelial cells. The method was validated using a SMC-specific lineage tracing mouse model wherein SM MHC Cre mice are crossed to ROSA flox STOP eYFP+/+ ApoE -/- mice. The H3K4dime/SM MHC PLA signal (i.e. PLA+) was exclusively found in eYFP+ cells. Moreover, some of the lesion SMCs were eYFP+/PLA+/SM α-actin-. Similarly, we identified PLA+ cells in human lesions that were positive for the macrophage marker CD68. Conclusion: Our new method permits definitive identification of SMC-derived cells within lesions even if they are not identifiable as SMC due to loss of SMC markers. Moreover, we provide exciting evidence that a significant fraction of macrophage-like cells in human lesions are derived from SMC. We postulate that transition of SMC to a macrophage state may be a key event leading to plaque destabilization and rupture with possible myocardial infarction or stroke.

Specification of secondary mesenchyme-derived cells in relation to the dorso-ventral axis in sea urchin blastulae.

To learn how the dorso-ventral (DV) axis of sea urchin embryos affects the specification processes of secondary mesenchyme cells (SMC), a fluorescent dye was injected into one of the macromeres of 16-cell stage embryos, and the number of each type of labeled SMC was examined at the prism stage. A large number of labeled pigment cells was observed in embryos in which the progeny of the labeled macromere were distributed in the dorsal part of the embryo. In contrast, labeled pigment cells were scarcely noticed when the descendants of the labeled macromere occupied the ventral part. In such embryos, free mesenchyme cells (probably blastocoelar cells) were predominantly labeled. CH3COONa treatment, which is known to increase the number of pigment cells, canceled such patterned specification of pigment cells and blastocoelar cells along the DV axis. Pigment cells were also derived from the ventral blastomere in the treated embryo. In contrast, a similar number of coelomic pouch cells was derived from the labeled macromere, irrespective of the position of its descendants along the DV axis. After examination of the arrangement of blastomeres in late cleavage stage embryos, it was determined that 17-20 veg2-derived cells encircled the cluster of micromere descendants after the 9th cleavage. From this number and the numbers of SMC-derived cells in later stage embryos, it was suggested that the most vegetally positioned veg2 descendants at approximately the 9th cleavage were preferentially specified to pigment and blastocoelar cell lineages. The obtained results also suggested the existence of undescribed types of SMC scattered in the blastocoele.

In situ screening for genes expressed preferentially in secondary mesenchyme cells of sea urchin embryos.

In sea urchin embryos, four types of non-skeletogenic mesodermal cells are derived from secondary mesenchyme cells (SMCs). Although determining the complete lineage of SMCs is currently a high-priority goal, specific markers for each type of SMC-derived cell in Hemicentrotus pulcherrimus are unavailable. To identify genes preferentially expressed in the various types of SMC-derived cells, we constructed a cDNA library of the archenteron isolated from late gastrulae. Both the 5' and 3' ends of 1,050 cDNAs randomly selected from 7,500 picked clones were sequenced. Based on the sequence at the 3' end, the cDNAs were grouped into 671 independent clusters. Among these, 605 clusters were analysed by whole-mount in situ hybridisation; 28% (170 clusters) exhibited differential expression patterns, while 24% were ubiquitously expressed and 48% did not show any staining. Of 170 clusters showing differential expression patterns, 33 clusters were differentially expressed in SMC-derived cells. From these clusters, several genes were obtained that were specifically or predominantly expressed in each type of SMCs, including coelomic pouch cells in which specific expression patterns have not been reported previously, and hence will be useful for lineage studies. Furthermore, in situ hybridisation revealed the existence of a new type or subpopulation of SMCs distributed sparsely in the blastocoel.