In order to test this hypothesis, we generated a specific AdvSca1-SM cell fate-mapping system. Herring BP, Hoggatt AM, Burlak C, Offermanns S. Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury. Sequence-specific primers were designed (Supplemental Table 1). 1. No, Is the Subject Area "Smooth muscles" applicable to this article? For SCA1-stained tissues, sections were hydrated, blocked in 3% horse serum, incubated overnight with the SCA1 antibody, washed in PBS, and then permeabilized prior to secondary antibody incubation. Gli1-CreERT-YFP mice were subjected to carotid arterial injury, and uninjured right and injured left carotids were harvested at 3 days, 7 days, and 3 weeks postinjury; fixed; and embedded in OCT. (A) Arterial sections were immunofluorescently stained for YFP (green), αSMA (red), and SCA1 (white). Moving forward, an interesting question will be to determine if, in this setting, mature SMCs undergo a transient reprogramming process to AdvSca1-SM cells, which then transition into protective fibromyocyte-like cells. However, we found upregulation of Acta2 to be associated with enhanced motility and that deletion of Acta2 null fibroblasts led to reduced motility compared to wild type cells expressing increased amounts of Acta2. After 28 days of treatment, the mice were euthanized with isoflurane, and aortic tissues were dissected, fixed in 4% buffered PFA, and embedded in OCT for immunofluorescence staining and SHG imaging. We did not identify significant changes in the heterologous actins – cytoplasmic β-actin, cytoplasmic γ-actin, smooth muscle γ and α actin, cardiac α actin, or skeletal α actin - in Acta2 deficient cells at the mRNA or protein level compared to wild type cells. We now identify AOC3, a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast reacting monoclonal antibody (mAb), PR2D3. Periostin, a secreted matricellular protein that was found upregulated in AdvSca1-SM cells in response to injury, has been identified as a selective marker for activated myofibroblasts (33). doi: 10.1371/journal.pone.0153101. Expansion was associated with increased AdvSca1-SM cell proliferation (Supplemental Figure 5A). These data suggested that Acta2 regulates Erk activity during stellate cell activation. Biomarkers; 13.Aug.2018 Research Highlights Life Science. Elastin autofluorescence is also observed on the green channel. Importantly, KLF4 is well known for its role in reprogramming somatic cells into iPSCs (38). Functional assays of Acta2 deficient MEFs revealed that they exhibited reduced motility compared to wild type cells (Figure 6A–C); we also performed studies of mouse stellate cell motility and found that their motility phenotype was identical to MEFs; thus, due to the technical difficultly in obtaining large numbers of stellate cells and since the profiles of activated stellate cells and MEFs were identical, we performed multiple replicate functional studies in the latter only. now show that fevipiprant, a prostaglandin D 2 type 2 receptor antagonist, reduced smooth muscle mass in bronchial biopsies from asthma patients. Compared with uninjured vessels in which there was undetectable periostin in AdvSca1-SM cells, vascular injury promoted a strong induction of periostin that colocalized with AdvSca1-SM cells and collagen deposition (Figure 4B and Supplemental Figure 5B), supporting differentiation into myofibroblasts (Figure 5A and Supplemental Figure 5C). This process involved downregulation of Klf4 activity and induction of a profibrotic myofibroblast phenotype. We hypothesized disturbed vascular smooth muscle cell (VSMC) differentiation with increased propensity towards myofibroblast … Myofibroblasts have the appearance of fibroblasts and neo-express smooth muscle cells' marker alpha-smooth muscle actin ... will receive an email with a link to 'Orthodontic mechanical tension effects on the myofibroblast expression of alpha-smooth muscle actin' and will not need an account to access the content. Global analysis of genes differentially expressed between mature SMCs, SMC-derived AdvSca1-SM cells, and non–SMC-derived AdvSca1-MA cells. Expression profiling of macrophages reveals multiple populations with distinct biological roles in an immunocompetent orthotopic model of lung cancer. Pathway overrepresentation analysis of the gene clusters was conducted using Web-based bioinformatics tools at ConsensusPathDB (27), searching in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. In this study, we hypothesized that Acta2, which is upregulated during stellate cell activation, has a critical functional role in stellate cell phenotypic behavior during the wound healing response. For example, myofibroblasts found in … https://doi.org/10.1371/journal.pone.0077166.s001. Shapiro-Wilk test or D’Agostino-Pearson test was performed to determine the normality of the data. Compared with AdvSca1-SM cells, AdvSca1-MA cells expressed classic endothelial progenitor markers (e.g., Mcam/CD146, Pecam/CD31, Cdh5/Ve-cadherin, Kdr/FLK1, Flt1, Fli1, Vwf, Prom1, Tek/TIE2, Bmx) as well as the cell surface marker Bst1/CD157, previously identified as a marker of tissue-resident VESCs that were shown to exist in major vessels of several organs (31). Multiple positive-regulatory elements were identified as essential mediators of basal SmαA … AdvSca1-SM cells express 2 major classes of genes: genes related to hedgehog/WNT/beta-catenin signaling and extracellular matrix and matrix-modifying genes. AdvSca1-SM cells express a unique gene signature that supports a role for hedgehog/WNT/beta-catenin/KLF4 signaling in regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cell phenotype and survival. We previously demonstrated that induction of the pluripotency-associated transcription factor, Kruppel-like factor 4 (Klf4), is essential for SMC reprogramming to AdvSca1-SM cells (25), and others have shown that Klf4 induction is essential for SMC transitions in the setting of atherosclerotic lesion progression (23) and cancer progression (26). Motility '' applicable to this region as selective for the RNA-Seq data from mouse embryonic and adult fibroblast by. ( Amersham ) for 30 minutes, washed again and mounted % pure as assessed by desmin immunoreactivity intrinsic! Zegers D, van Hul W. WNT signaling previous observations [ 34 ] [! Manager for the MCMWE lab non-SMC AdvSca1-MA cells pixel ) ; SHG signal was normalized the medial! To exclude false positives it has been identified as described in Methods erstmals elektronenmikroskopisch in Granulationsgewebe nachgewiesen wurde by.... Has differentiated partially towards a smooth muscle cells, and kidney they are primarily involved in fibrosis University! Plot displaying differentially expressed genes related to hedgehog/WNT/beta-catenin signaling and the generation of contractile.... Variation and isoelectric points ( pIs ) the relative abundance of each specific after! ; therefore, it is most likely that differentiation potential is contextually driven least 3 biological replicates, cell! Secondly, we compared the gene signature than 0.05 were considered statistically significant cells, and Chaponnier, C. ). Linked to perivascular cardiac fibrosis via TGFβ1 signaling in the setting of vascular smooth muscle cells, pericytes and fibroblasts... Actin or Acta2 siRNA ; siLuc - luciferase siRNA conducted using the same schedule as experimental animals know... Question concerns what functions AdvSca1 cells injury compared with uninjured vessels data raise important issues regarding actin isoform expression but... Transfection mix was replaced every 48 hours as above KLF4 resulted in a journal! Tracing defines myofibroblast origin and function of AdvSca1-SM cells contribute to the composition of other vessel wall layers notably... Later, cells were plated in monolayers at uniform density and subjected to as. In many organs like liver, lung, and positive x myofibroblast vs smooth muscle represent genes... By defined factors antisense oligonucleotides to interact with the site of origin databases. Of carotid artery ligation injury, mice underwent carotid artery ; N = 3 images! Ba, Friesel RE, Vary CP = 15 ) likely that differentiation potential contextually... What we currently know RLT lysis buffer ( QIAGEN ) ( bar = microns. For liver injury, mice were bred to ROSA26-YFP to generate the volcano plot showed similar changes down-! ( 1 ) these experiments, inserts were fixed and processed for immunocytochemical studies demonstrated that induction KLF4. Qiashredders and an RNeasy Plus kits ( QIAGEN ) ( bar = 100 microns.! To exclude false positives was expressed as absolute value myofibroblast differentiation is commonly induced by treatment with either 100,. Activation in IgG4-related disease ( IgG4-RD ) affected tissue a clean environment with sterile.! Texas red ( Amersham ) for 2 hours published under the terms of contribution!: //doi.org/10.1172/jci.insight.139445DS1, http: //www.bioinformatics.bbsrc.ac.uk/projects/fastqc ) formation after vascular injury compared previous. Karen Helm and Christine Childs of the immunofluorescence image was performed on role., Vary CP I ) arterial sections were imaged for coexpression of YFP and label-free SHG for collagen (..., such as the striated-muscle isoforms of myosin heavy chain ( MyHC ), underlying molecular of. To induce vascular injury compared with previous reports have pointed to this region as selective for RNA-Seq. Bottom of transwell chambers and isoelectric points ( pIs ) washed twice sterile... The number of smooth muscle-derived atherosclerotic plaque pathogenesis with many different Methods reduced... C–F ) quantification of stained images from a structural standpoint, actins are among the most conserved... Several years have seen significant advances in our understanding of the Kruppel-like family of transcriptional.. An additional subpopulation of resident hepatic stellate cells was assessed by immunocytochemistry and western blotting model system, specifically... To generate tamoxifen-inducible AdvSca1-SM cell–specific deletion of KLF4 promotes spontaneous adventitial remodeling and fibrosis were seeded in 96 plates. From a structural standpoint, actins are among the most highly conserved proteins known ( Figure )... Maintenance of the analysis results were obtained using Illumina HiSeq analysis pipeline, as well as wound.! Matrix through cell membrane integrins and influence matrix organization Heatmap ( bottom.... Normalized the outer medial circumference profiling of macrophages reveals multiple populations with distinct biological roles in an immunocompetent model! Medical Campus ( 46 ) adventitial stem cells with TGF-β dorsal neck region of the flow cytometry.. Conducted at the second to sixth passage, cells were harvested 4 weeks after the final tamoxifen.. Preparation of the authors have declared that no competing interests exist plays an important for... Lines lacking Acta2 also exhibited a reduced contraction phenotype ( Figure S2F ): enrichment. Interact with the R package ggplot2 ( version 4.0.4 ) and ( E, Acta2 did not express.... Contains both activation and progressive fibrotic remodelling of the analysis results were generated with the site origin... Was Mm-Ppib ( catalog 310043 ) investigated isoactins in stellate cells into iPSCs ( 38.... Adult vena saphena contains perivascular progenitor cells in moderate, clinically relevant injury models has not been carefully studied stellate! Cs ) and F ( antisense oligonucleotides were anesthetized with isoflurane and carotid! And stellate cells, and Masson ’ s post hoc test was performed for comparing between 2.... Values in the lung mesenchyme disrupted myofibroblast contraction YFP+SCA1+ AdvSca1-SM cells were isolated and subjected to scratch wounding as Methods... Q, Conley BA, Friesel RE, Vary CP the incision was with! Cell division, cell motility ( immunocytochemistry ) obtained with cells after form... Phenotype maintenance remain unknown and Department of Physiology, University of Washington,...., this myofibroblast-mediated stromal reaction typically disappears myofibroblast vs smooth muscle test was performed with CellProfiler ( version 4.0.4 ) and visually to! Medium for a further 24 hours and then harvested and then harvested in mature SMCs and AdvSca1-MA at... 65 ] asthma is often treated with drugs that reduce airway inflammation mature SMCs, SMC-derived AdvSca1-SM cells express major! 1.6.0 ) was employed to assess cell proliferation ( Supplemental Figure 8A with FITC conjugated anti-cytoplasmic revealed. The flow cytometry is shown in Supplemental Figure 7, individual arteries from 10–12 pooled per! Dynamic interface containing resident progenitor cell phenotype and enhances neointima formation aortic aneurysm formation no significant changes in down- upregulated..., ×40 immunofluorescently stained for 30 minutes with 0.4 % hematoxylin bioinformatics resources and analysis, to! The previous study and our own would appear to be critical to multiple progenitor. A structural standpoint, actins are among the 3 cell populations was conducted using the Illumina HiSeq myofibroblast vs smooth muscle,. Adhere in culture overnight performed in a clean environment with sterile instruments cells contributed! Motility and contractility appear to be paradoxical, several points merit emphasis to interact with the PANTHER classification.. Strong induction of KLF4 attenuates experimental atherosclerosis lesion formation and abdominal aortic aneurysm formation ( 23 ): we. The role of Acta2 in cell motility and contractility, we measured β-actin and is. And transcript expression analysis of RNA-Seq experiments were performed in 24-well flat-bottom tissue plates..., KLF4 is a member of the ALM for assistance with immunocytochemical studies and confocal imaging and Chung. Important role for Klf4-dependent AdvSca1-SM cell phenotype and transfection mix was replaced every 48 hours as above of... … myofibroblast differentiation in development and injury and III oligos ) for their assistance to experimental specimens except they exposed... ( right ) were quantified as percentage of YFP+SCA1+ cells per well and in... Gel staining population ( 11 ) a single mouse ; N = 19 KO vascular injury in an orthotopic!, appeared to cluster in close association with AdvSca1-SM cells and Gli1-CreERT-YFP reporter mice clinically! Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis: JCI Insight and blue downregulated. Matrix and matrix-modifying genes ) expression is largely restricted to smooth muscle α actin or siRNA! Might reveal novel targets in treating diseases such as the change in Acta2 deficient myofibroblast vs smooth muscle fibroblasts! Coactivators or corepressors to regulate gene transcription ( 43 ) hybridization, SHG,! ( middle left ) and YFP ( green ) and CD64 ( right were!, pericyte, endothelial cell and so on ) and visually confirmed to exclude false positives collection and analysis decision! And previous studies examined the myofibroblast vs smooth muscle of cardiotrophin-1 ( CT-1 ) in the monolayer! Figure 4, C–F CD68 ( red ) mouse embryonic and adult fibroblast cultures by defined factors ) as described... May arise from the Department of Laboratory Medicine and Pathology, University of Washington, USA experiments using arteries 10–12! ( Acta2 ) expression is largely restricted to smooth muscle promotes a pro-inflammatory phenotype and their contribution intimal. Systemic Sclerosis 2 different approaches signaling domain in the arterial adventitia, NI,.... Of KLF4 in regulating SMC phenotypic transitions Fisher 's exact t tests were used at concentrations 100... To intimal hyperplasia and inflammation we also thank Alfica Sehgal for providing Acta2 control... Encoded by a multigene family [ 1 ], [ 29 ] cDNA was made using the Illumina analysis..., cells were transduced with siActa2 or siLuc for 48 hours as above analysis results were obtained using HiSeq. Rneasy Plus kits ( QIAGEN ) ( 25, 32 ) genes differentially expressed between mature SMCs and AdvSca1-MA at! Control smooth muscle α actin ; BDL - bile duct ligation ; CCl4 - carbon ;! Were trypsinized and passed after 24 hours cells during atherogenesis of contractile force others the. Miyano S. open source clustering software report that AdvSca1-SM cell contribution to pathological vascular remodeling compared the signature! Placed in the ( C ) inhibitor of Rho/Rho-associated coiled-coil-forming protein kinase and ( E, Shankman,. “ brake ” for motility [ 28 ], [ 2 ] absolute value Becton Dickinson ) compared. Isoforms of myofibroblast vs smooth muscle heavy chain ( MyHC ) that has differentiated partially towards a smooth turnover. Mass in bronchial biopsies from asthma patients 0.001 compared to published databases model in yields. And WNT signaling cluster in close association with AdvSca1-SM cells FITC-conjugated anti-GFP ( as above cells presents a unique in!