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

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Laura S Shankman ◽  
Olga A Cherepanova ◽  
Delphine Gomez ◽  
Gary K Owens
Keyword(s):  
Smooth Muscle ◽  
Foam Cell ◽  
Lineage Tracing ◽  
Phenotypic Switching ◽  
Marker Genes ◽  
Plaque Stability ◽  
Pluripotency Factors ◽  
Fibrous Cap ◽  
Atherosclerotic Lesions ◽  
Life Threatening

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.

scholarly journals SMC Derived Hyaluronan Modulates Vascular SMC-Phenotype in Murine Atherosclerosis

2021 ◽  
Author(s):  
Felicia Hartmann ◽  
Daniel J Gorski ◽  
Alexandra AC Newman ◽  
Susanne Homann ◽  
Anne Petz ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transition State ◽  
Acute Phase ◽  
Acute Phase Response ◽  
Lineage Tracing ◽  
Phase Response ◽  
Phenotypic Switching ◽  
Plaque Stability ◽  
Specific Deletion

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 extracellular matrix (ECM) deposition. Objective: The aim of this study was to elucidate the role of hyaluronan (HA) derived from SMC-HA 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 (eYFP) on an Apoe-/- background was used. Lineage tracing of SMC with eYFP revealed that SMC-specific deletion of Has3 significantly increased the number of galectin-3 (LGALS3+) "transition-state" SMC and decreased alpha-smooth muscle actin (ACTA2+) SMC. Notably, SMC-Has3 deletion led to significantly increased collagen deposition and maturation within the fibrous cap (FC) and the whole lesion, as evidenced by Picrosirius red staining and LC-PolScope analysis. Single-cell RNA sequencing (scRNA-seq) of brachiocephalic artery (BCA) lesions demonstrated that the loss of SMC-Has3 enhanced the transition of SMC to an Lgals3+, ECM-producing phenotype with elevated acute-phase response gene expression. Experiments using cultured murine aortic SMC revealed that blocking cluster of differentiation-44 (CD44), an important HA 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.

scholarly journals Vascular smooth muscle cells in atherosclerosis: time for a re-assessment

2021 ◽  
Author(s):  
Mandy O J Grootaert ◽  
Martin R Bennett
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Atherosclerotic Lesion ◽  
Muscle Cells ◽  
Lineage Tracing ◽  
Phenotypic Switching ◽  
Fibrous Cap ◽  
The Media

Abstract Vascular smooth muscle cells (VSMCs) are key participants in both early and late-stage atherosclerosis. VSMCs invade the early atherosclerotic lesion from the media, expanding lesions, but also forming a protective fibrous cap rich in extracellular matrix to cover the ‘necrotic’ core. Hence, VSMCs have been viewed as plaque-stabilizing, and decreased VSMC plaque content—often measured by expression of contractile markers—associated with increased plaque vulnerability. However, the emergence of lineage-tracing and transcriptomic studies has demonstrated that VSMCs comprise a much larger proportion of atherosclerotic plaques than originally thought, demonstrate multiple different phenotypes in vivo, and have roles that might be detrimental. VSMCs down-regulate contractile markers during atherosclerosis whilst adopting alternative phenotypes, including macrophage-like, foam cell-like, osteochondrogenic-like, myofibroblast-like, and mesenchymal stem cell-like. VSMC phenotypic switching can be studied in tissue culture, but also now in the media, fibrous cap and deep-core region, and markedly affects plaque formation and markers of stability. In this review, we describe the different VSMC plaque phenotypes and their presumed cellular and paracrine functions, the regulatory mechanisms that control VSMC plasticity, and their impact on atherogenesis and plaque stability.

Abstract 512: The Long Non-coding Rna MIAT Regulates Smooth Muscle Cell Proliferation and Macrophage Activity in Advanced Atherosclerotic Lesions

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Yuhuang Li ◽  
Hong Jin ◽  
Ljubica Perisic ◽  
Ekaterina Chernogubova ◽  
Alexandra Bäcklund ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Oxidized Ldl ◽  
In Silico Analysis ◽  
Human Monocyte ◽  
Carotid Plaques ◽  
Knock Down ◽  
Plaque Stability ◽  
Atherosclerotic Lesions ◽  
Markers Of Inflammation

Background: Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators in various biological processes and diseases. Here we sought to identify and functionally characterize the lncRNA MIAT as a novel regulator in atherosclerotic plaque stability. Methods and results: We profiled RNA transcript expression in patients with advanced atherosclerotic lesions from the Biobank of Karolinska Endarterectomies (BiKE). By microarray analysis, lncRNA MIAT was identified as one of the most highly up-regulated non-coding RNAs in carotid plaques compared to iliac artery controls, which was confirmed by qRT-PCR and in situ hybridization. Additional in silico analysis indicated a substantial positive correlation of MIAT with markers of inflammation, apoptosis and matrix degradation in carotid plaques. Experimental knock-down of MIAT, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) not only markedly decreased proliferation and migration rates of cultured human carotid artery smooth muscle cells (hCASMCs), but also increased their levels of apoptosis. In addition, MIAT inhibition significantly impaired oxidized LDL (oxLDL) uptake of murine peritoneal as well as human monocyte-differentiated macrophages in vitro. In contrast, induction of MIAT expression by lipoprotein-a (LPa) treatment, displayed the opposite effect. Conditioned medium from macrophage cultures after MIAT knock-down substantially decreased hCASMC proliferation, indicating a potential involvement of MIAT in macrophage-SMC interactions during advanced stages of atherosclerosis. Conclusion: The lncRNA MIAT is a novel regulator of cellular processes in atherosclerosis and plaque stability, which influences SMC proliferation and apoptosis and interacts with disease-triggering macrophages.

scholarly journals Krüppel-like factor 4, Elk-1, and histone deacetylases cooperatively suppress smooth muscle cell differentiation markers in response to oxidized phospholipids

2008 ◽  
Vol 295 (5) ◽  
pp. C1175-C1182 ◽  
Author(s):  
Tadashi Yoshida ◽  
Qiong Gan ◽  
Gary K. Owens
Keyword(s):  
Smooth Muscle ◽  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Phenotypic Switching ◽  
Marker Genes ◽  
Physical Interaction ◽  
Oxidized Phospholipids ◽  
Differentiation Markers ◽  
Differentiation Marker

Phenotypic switching of vascular smooth muscle cells (SMCs), such as increased proliferation, enhanced migration, and downregulation of SMC differentiation marker genes, is known to play a key role in the development of atherosclerosis. However, the factors and mechanisms controlling this process are not fully understood. We recently showed that oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)- sn-glycero-3-phosphocholine (POVPC), which accumulate in atherosclerotic lesions, are potent repressors of expression of SMC differentiation marker genes in cultured SMCs as well as in rat carotid arteries in vivo. Here, we examined the molecular mechanisms whereby POVPC induces suppression of SMC differentiation marker genes in cultured SMCs. Results showed that POVPC induced phosphorylation of ERK1/2 and Elk-1. The MEK inhibitors U-0126 and PD-98059 attenuated POVPC-induced suppression of smooth muscle ( SM) α-actin and SM-myosin heavy chain. POVPC also induced expression of Krüppel-like factor 4 (Klf4). Chromatin immunoprecipitation assays revealed that POVPC caused simultaneous binding of Elk-1 and Klf4 to the promoter region of the SM α-actin gene. Moreover, coimmunoprecipitation assays showed a physical interaction between Elk-1 and Klf4. Results in Klf4-null SMCs showed that blockade of both Klf4 induction and Elk-1 phosphorylation completely abolished POVPC-induced suppression of SMC differentiation marker genes. POVPC-induced suppression of SMC differentiation marker genes was also accompanied by hypoacetylation of histone H4 at the SM α-actin promoter, which was mediated by the recruitment of histone deacetylases (HDACs), HDAC2 and HDAC5. Coimmunoprecipitation assays showed that Klf4 interacted with HDAC5. Results provide evidence that Klf4, Elk-1, and HDACs coordinately mediate POVPC-induced suppression of SMC differentiation marker genes.

scholarly journals Multiple repressor pathways contribute to phenotypic switching of vascular smooth muscle cells

2007 ◽  
Vol 292 (1) ◽  
pp. C59-C69 ◽  
Author(s):  
Keiko Kawai-Kowase ◽  
Gary K. Owens
Keyword(s):  
Smooth Muscle ◽  
Molecular Mechanisms ◽  
Platelet Derived Growth Factor ◽  
Phenotypic Switching ◽  
Marker Genes ◽  
Environmental Cues ◽  
Selective Marker ◽  
Current State ◽  
Active Repressor

Smooth muscle cell (SMC) differentiation is an essential component of vascular development and these cells perform biosynthetic, proliferative, and contractile roles in the vessel wall. SMCs are not terminally differentiated and possess the ability to modulate their phenotype in response to changing local environmental cues. The focus of this review is to provide an overview of the current state of knowledge of molecular mechanisms involved in controlling phenotypic switching of SMC with particular focus on examination of processes that contribute to the repression of SMC marker genes. We discuss the environmental cues which actively regulate SMC phenotypic switching, such as platelet-derived growth factor-BB, as well as several important regulatory mechanisms required for suppressing expression of SMC-specific/selective marker genes in vivo, including those dependent on conserved G/C-repressive elements, and/or highly conserved degenerate CArG elements found in the promoters of many of these marker genes. Finally, we present evidence indicating that SMC phenotypic switching involves multiple active repressor pathways, including Krüppel-like zinc finger type 4, HERP, and ERK-dependent phosphorylation of Elk-1 that act in a complementary fashion.

2007 Russell Ross Memorial Lectureship in Vascular Biology—Epigenetic Control of Vascular Smooth Muscle Differentiation in Development and Disease

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Gary K. Owens
Keyword(s):  
Smooth Muscle ◽  
Vascular Injury ◽  
Serum Response Factor ◽  
Critical Role ◽  
Conditional Knockout ◽  
Phenotypic Switching ◽  
Marker Genes ◽  
List Type ◽  
Response Factor ◽  
Serum Response

There is clear evidence that alterations in the differentiated state of the smooth muscle cell (SMC) play a key role in the pathogenesis of a number of major human diseases, including atherosclerosis and postan-gioplasty restenosis. This process is referred to as “phenotypic switching” and likely evolved to promote repair of vascular injury. However, the mechanisms controlling phenotypic switching as well as normal differentiation of SMCs in vivo are poorly understood. This talk will provide an overview of molecular mechanisms that control differentiation of SMCs during vascular development. A particular focus will be to consider the role of CArG elements found within the promoters of many SMC differentiation marker genes, as well as regulation of their activity by serum response factor and the potent SMC-selective serum response factor coactivator myocardin. In addition, I will summarize recent work in our laboratory showing that SMC- and gene-locus–selective changes in chromatin structure play a critical role both in normal control of SMC differentiation and in phenotypic switching in response to vascular injury. Finally, I will present evidence based on conditional knockout experiments in mice showing that krupple-like factor 4 is induced in SMCs after vascular injury and regulates SMC phenotypic switching and growth through: binding to G/C repressor elements located in close proximity of CArG elements within the promoters of many SMC marker genes, suppressing expression of myocardin, and inducing epigenetic modifications of SMC marker gene loci associated with chromatin condensation and transcriptional silencing. Supported by NIH grants P01 HL19242, R37 HL57353, and R01 HL 38854.

Abstract 17038: Twist1 Modulates Smooth Muscle Cell Phenotypes in Atherosclerosis

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Marie A Guerraty ◽  
Sylvia T Nurnberg ◽  
Vraj Shah ◽  
Daniel J Rader
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Risk Allele ◽  
Muscle Cells ◽  
Chromosome 9 ◽  
Alizarin Red ◽  
Plaque Stability ◽  
Atherosclerotic Lesions ◽  
Mesenchymal Transformation

Introduction: Genome-wide association studies have identified rs2107595, a non-coding locus on chromosome 9 between HDAC9 and Twist1 genes, as a risk allele for several vascular phenotypes, including Coronary Artery Disease (CAD). Rs2107595 has, more specifically, been associated with stable CAD over myocardial infarction phenotypes. Recent work has shown that rs2107595 risk allele increases Twist1 expression in smooth muscle cells (SMCs) by creating an RBPJ binding site. In other cell types, Twist1 is known to maintain cells in a de-differentiated state and to promote epithelial to mesenchymal transformation, driving tumor progression and metastasis. Hypothesis: Twist1 modulates SMC differentiation to promote an immature proliferative state over a differentiated (osteoblastic) state. This shift in phenotype promotes features of plaque stability in vivo . Methods: Twist1 expression plasmid (pCMV6-TWIST1) was transfected into A7r5 rat smooth muscle cells. To assess proliferation, cells were counted at 24, 48, 72, and 96 hours. To assess calcification, A7r5 cells were cultured in calcification media (2mM NaPhos) for 10 days and stained with Alizarin Red. In vivo studies were performed in Twist1 fl/fl tamoxifen-inducible MYH11-Cre C57BL/6 mice on ApoE-/- background fed a Western diet for 16 weeks to induce atherosclerotic lesions. Immunohistochemistry with SM22a identified lesion SMCs, and alizarin red was used to identify calcifications. Results: Ectopic overexpression of Twist1 in A7r5 SMCs decreased proliferation at 48h and 72h (80%, p=0.014). Twist 1 overexpression also decreased the total area of calcification (33% reduction, p=0.007). In vivo , both control and Twist 1 KO mice show similar burden of atherosclerosis. However, there is a decrease in sub-endothelial SMCs in atherosclerotic lesions by SM22a staining in the Twist1 KO. Additionally, Twist1 KO mice have more prominent and larger focal calcifications. Conclusions: Twist1 promotes SMC proliferation and decreases calcification in vitro , and may affect the presence of subendothelial SMCs and calcification in vivo . This provides a compelling link that rs2107595 may promote plaque stability in CAD by increasing Twist1 to modulate SMC phenotypes.

scholarly journals Smooth Muscle Overexpression of PGC1α Attenuates Atherosclerosis in Rabbits

2021 ◽  
Author(s):  
Zhe Wei ◽  
Hoshun Chong ◽  
Qixia Jiang ◽  
Yuhang Tang ◽  
Jinhong Xu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Plasma Cholesterol ◽  
Critical Role ◽  
Phenotypic Switching ◽  
Marker Genes ◽  
High Cholesterol Diet ◽  
Contractile Phenotype ◽  
Promising Therapeutic Approach

Rationale: Targeting vascular smooth muscle cell (VSMC) phenotypic switching is a promising therapeutic approach for atherosclerosis (AS). Dysregulation of PGC1α, a key regulator of cellular energy metabolism, has been implicated in the pathogenesis of AS, yet its role in AS remains controversial. Objective: The current study aimed to determine whether and how PGC1α in VSMCs regulates AS progression. Methods and Results: We generated transgenic (Tg) rabbits with SMC-specific PGC1α overexpression and showed that these rabbits developed significantly less aortic AS than their non-Tg littermates after high-cholesterol diet (HCD) feeding, while total plasma cholesterol levels were similar. As indicated by the restored expression of VSMC differentiation marker genes, the HCD-induced phenotypic switching in the aortic media was largely reversed in Tg rabbits, accompanied by decreased levels of synthetic phenotype genes, proinflammatory cytokines, adhesion molecules, macrophage infiltration, matrix metalloproteinases (MMPs), reactive oxygen species (ROS) production and senescence. Ex vivo studies further showed that VSMC-specific PGC1α overexpression markedly suppressed the promotive effect of HCD feeding on the association of serum response factor (SRF) with ELK1, a ternary complex factor (TCF) that acts as a myogenic repressor in VSMCs, thereby preserving the VSMC contractile phenotype. Furthermore, knockdown of PGC1α remarkably increased extracellular signal-regulated kinase (ERK)1/2-ELK-1 signaling, which promoted phenotypic switching and proliferation of cultured rabbit VSMCs. In addition, we showed that PGC1α can regulate EGFR-ERK1/2 MAP kinase signaling via modulating PPARγ activity in RVSMCs. Finally, we showed that these beneficial results of SMC-specific PGC1α overexpression can be extrapolated from rabbits to human VSMCs and clinical settings. Conclusions: We demonstrated a critical role of PGC1α in maintaining the contractile phenotype of VSMCs and highlighted the therapeutic potential of PGC1α for AS.

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

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Delphine Gomez ◽  
Laura Shankman ◽  
Gary K Owens
Keyword(s):  
Smooth Muscle ◽  
Marker Gene ◽  
Atherosclerotic Lesion ◽  
Cell Types ◽  
Lineage Tracing ◽  
Phenotypic Switching ◽  
Proximity Ligation Assay ◽  
Mhc Locus

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.

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