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.

scholarly journals Lineage Tracing Reveals the Dynamic Contribution of Pericytes to the Blood Vessel Remodeling in Pulmonary Hypertension

2020 ◽  
Vol 40 (3) ◽  
pp. 766-782 ◽  
Author(s):  
Jennifer Bordenave ◽  
Ly Tu ◽  
Nihel Berrebeh ◽  
Raphaël Thuillet ◽  
Amélie Cumont ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Structural Changes ◽  
Chronic Hypoxia ◽  
Transforming Growth Factor ◽  
Primary Cultures ◽  
Lineage Tracing ◽  
In Vivo Model ◽  
Dependent Manner

Objective: Excessive accumulation of resident cells within the pulmonary vascular wall represents the hallmark feature of the remodeling occurring in pulmonary arterial hypertension (PAH). Furthermore, we have previously demonstrated that pulmonary arterioles are excessively covered by pericytes in PAH, but this process is not fully understood. The aim of our study was to investigate the dynamic contribution of pericytes in PAH vascular remodeling. Approach and Results: In this study, we performed in situ, in vivo, and in vitro experiments. We isolated primary cultures of human pericytes from controls and PAH lung specimens then performed functional studies (cell migration, proliferation, and differentiation). In addition, to follow up pericyte number and fate, a genetic fate-mapping approach was used with an NG2CreER;mT/mG transgenic mice in a model of pulmonary arteriole muscularization occurring during chronic hypoxia. We identified phenotypic and functional abnormalities of PAH pericytes in vitro, as they overexpress CXCR (C-X-C motif chemokine receptor)-7 and TGF (transforming growth factor)-βRII and, thereby, display a higher capacity to migrate, proliferate, and differentiate into smooth muscle-like cells than controls. In an in vivo model of chronic hypoxia, we found an early increase in pericyte number in a CXCL (C-X-C motif chemokine ligand)-12-dependent manner whereas later, from day 7, activation of the canonical TGF-β signaling pathway induces pericytes to differentiate into smooth muscle-like cells. Conclusions: Our findings reveal a pivotal role of pulmonary pericytes in PAH and identify CXCR-7 and TGF-βRII as 2 intrinsic abnormalities in these resident progenitor vascular cells that foster the onset and maintenance of PAH structural changes in blood lung vessels.

scholarly journals MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro

2010 ◽  
Vol 189 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Manuela Quintavalle ◽  
Leonardo Elia ◽  
Gianluigi Condorelli ◽  
Sara A. Courtneidge
Keyword(s):  
Smooth Muscle ◽  
Cell Types ◽  
Platelet Derived Growth Factor ◽  
Pdgf Receptor ◽  
Kinase C ◽  
Membrane Protrusions ◽  
Synthetic Phenotype ◽  
Single Promoter

Smooth muscle cell (SMC) plasticity plays an important role during development and in vascular pathologies such as atherosclerosis and restenosis. It was recently shown that down-regulation of microRNA (miR)-143 and -145, which are coexpressed from a single promoter, regulates the switch from contractile to synthetic phenotype, allowing SMCs to migrate and proliferate. We show in this study that loss of miR-143/145 in vitro and in vivo results in the formation of podosomes, which are actin-rich membrane protrusions involved in the migration of several cell types, including SMCs. We further show that platelet-derived growth factor (PDGF) mediates podosome formation in SMCs through the regulation of miR-143/145 expression via a pathway involving Src and p53. Moreover, we identify key podosome regulators as targets of miR-143 (PDGF receptor α and protein kinase C ε) and miR-145 (fascin). Thus, dysregulation of the miR-143 and -145 genes is causally involved in the aberrant SMC plasticity encountered during vascular disease, in part through the up-regulation of an autoregulatory loop that promotes podosome formation.

scholarly journals Human Cytomegalovirus Reduces Endothelin-1 Expression in Both Endothelial and Vascular Smooth Muscle Cells

2021 ◽  
Vol 9 (6) ◽  
pp. 1137
Author(s):  
Koon-Chu Yaiw ◽  
Abdul-Aleem Mohammad ◽  
Chato Taher ◽  
Huanhuan Leah Cui ◽  
Helena Costa ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Human Cytomegalovirus ◽  
Muscle Cells ◽  
Opportunistic Pathogen ◽  
Cell Types ◽  
Precursor Protein ◽  
Endothelin 1

Human cytomegalovirus (HCMV) is an opportunistic pathogen that has been implicated in the pathogenesis of atherosclerosis. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is overexpressed and strongly associated with many vasculopathies. The main objective of this study was to investigate whether HCMV could affect ET-1 production. As such, both endothelial and smooth muscle cells, two primary cell types involved in the pathogenesis of atherosclerosis, were infected with HCMV in vitro and ET-1 mRNA and proteins were assessed by quantitative PCR assay, immunofluorescence staining and ELISA. HCMV infection significantly decreased ET-1 mRNA and secreted bioactive ET-1 levels from both cell types and promoted accumulation of the ET-1 precursor protein in infected endothelial cells. This was associated with inhibition of expression of the endothelin converting enzyme-1 (ECE-1), which cleaves the ET-1 precursor protein to mature ET-1. Ganciclovir treatment did not prevent the virus suppressive effects on ET-1 expression. Consistent with this observation we identified that the IE2-p86 protein predominantly modulated ET-1 expression. Whether the pronounced effects of HCMV in reducing ET-1 expression in vitro may lead to consequences for regulation of the vascular tone in vivo remains to be proven.

scholarly journals Oxidized Phospholipids Induce Phenotypic Switching of Vascular Smooth Muscle Cells In Vivo and In Vitro

2007 ◽  
Vol 101 (8) ◽  
pp. 792-801 ◽  
Author(s):  
Nataliya A. Pidkovka ◽  
Olga A. Cherepanova ◽  
Tadashi Yoshida ◽  
Matthew R. Alexander ◽  
Rebecca A. Deaton ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Phenotypic Switching ◽  
Oxidized Phospholipids

scholarly journals OR03-02 Identification of a Novel Stem/Progenitor Population of the Adrenal Medulla

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Alice Santambrogio ◽  
John P Russell ◽  
Emily J Lodge ◽  
Laura D Scriba ◽  
Ilona Berger ◽  
...  
Keyword(s):  
Adrenal Medulla ◽  
Chromaffin Cells ◽  
Cell Types ◽  
Lineage Tracing ◽  
Genetic Lineage ◽  
Outer Cortex ◽  
Differentiation Potential ◽  
Sustentacular Cells

Abstract The adrenal glands regulate multiple physiological processes including the stress response, the immune system and metabolism. The adrenal is composed of an outer cortex that produces steroids, and an inner medulla that produces catecholamines. Tissue-specific stem/progenitor populations have been identified in the adrenal cortex, while the presence of a functional stem/progenitor population in the adrenal medulla is unclear. The adrenal medulla derives from the neural crest and contains chromaffin cells, neurons and sustentacular (support) cells. Establishing cell hierarchy and elucidating mechanisms of regulation of the different cell types is important to understand normal homeostasis and disease pathogenesis, such as of pheochromocytomas. Using genetic approaches in mouse, we have established that a subpopulation of sustentacular cells express the stem/progenitor marker SOX2. Through genetic lineage-tracing using the Sox2-CreERT2 strain, we demonstrate that these are an expanding population, capable of giving rise to chromaffin cells and neurons throughout life, consistent with a stem/progenitor role in vivo. We further demonstrate the self-renewal and differentiation potential of SOX2+ cells through in vitro isolation and expansion. Through analysis of FFPE sections of human adrenals, we confirm the presence of SOX2+ cells in the normal adult organ, as well as in pheochromocytomas. Taken together, our data support the identification of a previously undescribed stem/progenitor cell in the mammalian adrenal medulla, and confirm its functional relevance.

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.

scholarly journals PERK inhibition mitigates restenosis and thrombosis - a potential low-thrombogenic anti-restenotic paradigm

2019 ◽  
Author(s):  
Bowen Wang ◽  
Mengxue Zhang ◽  
Go Urabe ◽  
Guojun Chen ◽  
Debra Wheeler ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Ex Vivo ◽  
Cell Types ◽  
Brdu Incorporation ◽  
Main Stream ◽  
Stream Management ◽  
Drug Eluting ◽  
Normal Blood Flow

AbstractBackgroundDrug-eluting stents (DES) represent the main-stream management of restenosis following treatments of occlusive cardiovascular diseases. However, DES cannot eliminate instent restenosis yet exacerbate thrombogenic risks. To achieve dual inhibition of restenotic smooth muscle cell (SMC) de-differentiation/proliferation and thrombogenic endothelial cell (EC) dysfunction, a common target in both cell types, has been long-sought after. We evaluated the potential of protein kinase RNA-like endoplasmic reticulum kinase (PERK) as such a target for low-thrombogenic anti-restenotic intervention.Methods and ResultsWe used a rat angioplasty model of restenosis and a FeCl3-induced mouse model of thrombosis. Loss-or gain-of-function was achieved by PERK inhibition (GSK2606414, siRNA) or overexpression (adenovirus). Restenosis was robustly mitigated by GSK2606414 administered either via injected (i.v.) lesion-homing platelet membrane-coated nanoclusters or a perivascular hydrogel; it was enhanced by PERK transgene. Whereas PERK inhibition blocked, its overexpression exacerbated PDGF-induced human aortic SMC de-differentiation (reduced smooth muscle α-actin or αSMA) and proliferation (BrdU incorporation). Further, PERK activity promoted STAT3 activation but inhibited SRF transcriptional (luciferase) activity; its protein co-immunoprecipitated with STAT3 and also MRTF-A, the SRF activator for αSMA transcription. Importantly, PERK inhibition also prevented TNFα-induced impairment of human EC growth and upregulation of thrombogenic tissue factor, both in vitro and ex vivo. In vivo, oral gavage of GSK2606414 preserved ~50% of the normal blood flow 60 min after FeCl3-induced vascular injury.ConclusionsPERK inhibition is dual beneficial in mitigating restenosis and thrombosis, thus implicating a potential design for anti-restenotic intervention to overcome the thrombogenicity of DES.

scholarly journals Clonally expanding smooth muscle cells promote atherosclerosis by escaping efferocytosis and activating the complement cascade

2020 ◽  
Vol 117 (27) ◽  
pp. 15818-15826 ◽  
Author(s):  
Ying Wang ◽  
Vivek Nanda ◽  
Daniel Direnzo ◽  
Jianqin Ye ◽  
Sophia Xiao ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Immune Surveillance ◽  
Relative Survival ◽  
Inflammatory Cells ◽  
Muscle Cells ◽  
Lineage Tracing ◽  
Genomic Analyses

Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of “dedifferentiated” vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don’t eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.

scholarly journals Sp1-dependent activation of KLF4 is required for PDGF-BB-induced phenotypic modulation of smooth muscle

2009 ◽  
Vol 296 (4) ◽  
pp. H1027-H1037 ◽  
Author(s):  
Rebecca A. Deaton ◽  
Qiong Gan ◽  
Gary K. Owens
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Smooth Muscle ◽  
Vascular Injury ◽  
Marker Gene ◽  
Negative Regulator ◽  
Phenotypic Modulation ◽  
Marker Gene Expression

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.

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