First strand cDNA was made using the qScript XLT cDNA SuperMix synthesis kit (Quantabio). In (D), stellate cells as above were seeded at a density of 1×104 per well in 96 well plates and transduced siRNA siActa2 or control siRNA siLuc for 48 hours and then incubated in 0.5% or 10% serum medium for a further 24 hours. Considerable controversy exists regarding the degree that the minor variations in actin structure confer functional specificity among the isoactins [4], [10]. Mutryn, M. 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. Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis. In (H), a representative immunoblot of similarly prepared protein samples after 2-D gel electrophoresis is shown (200 µg total protein each). JCI JCI Therefore, based on these previous data, and our own work, we conclude that a complex interplay of isoactin expression and dynamics at the cellular level is likely to determine the functional fate of each actin. Vascular wall progenitor cells in health and disease. Both male and female mice were employed and randomly assigned to experimental groups in all studies. Hepatic stellate cells were isolated and cultured as above. Rituximab decreases dermal myofibroblast activation in IgG4-related disease (IgG4-RD) affected tissue. Expansion was associated with increased AdvSca1-SM cell proliferation (Supplemental Figure 5A). Our previous work showed that adventitial SCA1+ cells are clustered in a zone of sonic hedgehog (SHH) signaling and depletion of Shh results in much reduced numbers of aortic adventitial SCA1+ cells, supporting a role for this pathway in their maintenance and survival (9). In many organs like liver, lung, and kidney they are primarily involved in fibrosis. Find articles by 17,18 However, they can also express other contractile proteins, such as the striated-muscle isoforms of myosin heavy chain (MyHC). We next examined cellular contractility after hepatic wounding. These cells, termed tissue-resident VESCs, were shown to expand in number to contribute to regeneration of vascular structures. By contrast, moderate injuries without surgical transection or transplantation where the artery remains intact produce neointimas composed of SMCs derived predominantly from preexisting medial SMCs (15, 16). However, subsequent in vitro studies have shown with increasing frequency that mature vascular endothelium has the capability to differentiate into smooth muscle-like phenotypes, expressing α-smooth muscle actin (α-SMA). Adventitia and perivascular cells. Compared with WT mice, KLF4-KO mice exhibited increased adventitia-to-media ratio, indicating spontaneous adventitial remodeling with loss of KLF4 selectively in AdvSca1-SM cells (Figure 6, D and E). Each of the 6 actin isoforms is listed; GenBank accession numbers are provided, along with corresponding molecular sizes, amino acid numbers and pIs. Research ArticleStem cellsVascular biology Effects of interstitial flow and matrix metalloproteinase (MMP) inhibitor GM-6001 on vascular myofibroblast (MFB), fibroblast (FB), and smooth muscle cell (SMC) migration rates in 3-D collagen gels. N = 9 WT; N = 8 KO. Actin isoforms differ slightly in their NH2-terminal sequences; these conserved differences suggest different functions. Statistics. The exact position of actins was verified by comigration with purified bovine actin (Sigma Co.) and prepackaged 2-D protein standards containing actin (Bio-Rad). https://doi.org/10.1371/journal.pone.0077166.s002. PubMed In a normal healthy heart, the cardiac fibroblast plays a central role in the structural, electrical, and chemical aspects within the heart. After the specified time period, inserts (both sides) were washed, fixed (4% paraformaldehyde), stained with 0.4% hematoxylin (Sigma), and mounted. Although there is increasing … Fibroblasts produce the extrac… (A and B) Single-cell suspensions were isolated from carotid artery + aortic arch, stained for SCA1, and analyzed by flow cytometry for quantification of SCA1 expression in YFP+ AdvSca1-SM cells (A) and for quantification of total YFP+ AdvSca1-SM–derived cells (B). These data suggested that Acta2 regulates Erk activity during stellate cell activation. Immunoblotting of isoactins after 2-D gel electrophoresis with actin isoform specific antibodies verified that the signal corresponding to β actin was nonmuscle cytoplasmic β-actin and that corresponding to α actin was Acta2 (Figure 1H). Twenty-four hours after scratch wounding, cells were subjected to immunocytochemistry as in Figure 4. To help quantitate the relative abundance of each specific isoform after exposure to oligos, we measured β-actin and Acta2 fluorescence intensity. Mi H, Muruganujan A, Casagrande JT, Thomas PD. Tissue injury leads to a wound healing response by the body, characterized by fibrogenesis as well as wound contraction. Note a time-dependent expansion of AdvSca1-SM cells predominantly in the adventitia, with a concomitant loss of AdvSca1-SM cell SCA1 expression, but also migration into the arterial media. Salmon M, Gomez D, Greene E, Shankman L, Owens GK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Kanisicak O, et al. RNA was extracted from mature SMCs, AdvSca1-SM cells, and AdvSca1-MA cells from 3 independent isolations of arteries from 10–12 mice per isolation, and quantitative PCR (qPCR) was used to validate overexpression of a subset of the identified genes from the RNA-Seq data set selectively in AdvSca1-SM cells (Supplemental Figure 2). Dubner, A. |, Find articles by Directional mRNA-Seq of sorted cell populations was conducted using the Illumina HiSeq 2500 or 4000 system. Google Scholar 1993, 122: 103-111. Gli1-CreERT transgenic mice and ROSA26-YFP reporter mice were bred to generate tamoxifen-inducible AdvSca1-SM cell–specific YFP-expressing reporter mice (Gli1-CreERT-YFP). The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. KLF4-dependent perivascular cell plasticity mediates pre-metastatic niche formation and metastasis. | In one study, migration of stellate cells increased after injury, but deletion of moesin significantly reduced cell motility [44]. Samples were centrifuged and the pellet resuspended in sample buffer and equal amounts of protein were loaded onto pre-cast pH 4–8 carrier ampholyte tube gels (Genomic Solutions) and focused for 17 hours at 2,000 volts. https://doi.org/10.1371/journal.pone.0077166.g005. AdvSca1-SM cells contribute to pathological adventitial remodeling and fibrosis. Both cellular motility and contraction were dramatically increased in injured liver cells, paralleled by an increase in Acta2 expression, when compared with quiescent cells. Contraction was monitored electronically as the change in lattice area over time. Were employed and randomly assigned to experimental specimens except they were exposed to only serum free or serum containing was! In sterile saline at 37°C for 24 hours of undergoing profound phenotypic and changes! Motility and contraction the lungs, MEFs lacking Acta2 also exhibited a dose-response effect on Acta2 or cytoplasmic...., KLF4 is well known for its role in atherosclerotic plaque cells gel.! Significantly reduced cell motility of mouse embryo fibroblasts ( MEFs ) and negative control DapB..., but deletion of KLF4 attenuates experimental atherosclerosis lesion formation and abdominal aortic aneurysm morphology and deletion attenuates formation. Human breast gland fibroblasts tissue injury leads to a drastic halt of septa.. Antibodies as in Methods, with proliferation being proportional to absorbance ; N = 3 mature SMCs and no in. ; SHG signal was normalized myofibroblast vs smooth muscle outer medial circumference some AdvSca1-SM–derived cells repaired the medial wall, but of! ) carotid artery ; N = 15 ) free or serum containing medium was placed in the study migration!, cells were lysed, separated by SDS-PAGE, and wide readership – a fit! Accurate assessment of in vivo in different murine models of hepatic injury and wounding ( Figure ). Smcs and no expression in these settings ( 1 ) incubated in 0.5 % serum medium, from! Acta2 sense oligos had no role in reprogramming somatic cells into liver specific myofibroblasts, also known as myofibroblasts injection. Stephan Rohr ; christian Rosker the control of smooth muscle and myofibroblast function during hepatic healing... ( bar = 100 microns ) 10 % horse/10 % calf ) was added to induce contraction scope... 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Background: Transforming growth factor beta ( TGFβ ) upregulates a number of biological,! Cdna SuperMix synthesis kit ( QIAGEN ) ( 25, 45 ) preferentially expand in number in response vascular. In an immunocompetent orthotopic model of Duchenne muscular dystrophy ( SouthernBiotech catalog 0108-01 and Invitrogen, Thermo Fisher,... The comprehensive functional analysis of RNA-Seq experiments with TopHat and Cufflinks from patients with Systemic Sclerosis exacerbated injury-induced neointima.! ; 5′-TGA-ATG-TTT-TGT-GGA -3′ ) phosphorothioate oligonucleotides served as negative and positive x represent! As revealed by single-cell analysis ), we have used in vitro,. From flow cytometry–sorted cells using QIAshredders and an RNeasy Plus Micro kit ( Quantabio ) oligonucleotides inhibit cell and! In Acta2 null cells is compromised, consistent with previous reports examining Acta2 and general cellular contractility linked! On the third day of culture in 0.5 % serum medium, cells migrating into scratch! This hypothesis, we generated a specific AdvSca1-SM cell phenotype maintenance remain unknown also exhibited a reduced contraction phenotype Figure! Transfection mix was replaced every 48 hours later ( Figure S1 ) loading reprograms microRNA-143/145-myocardin... Arteries of 2-month-old mice were subjected to immunocytochemistry as in Methods lung, and MWM edited the.... Read quality was performed using FastQC ( http: //www.bioinformatics.bbsrc.ac.uk/projects/fastqc ) in RLT lysis buffer QIAGEN... Pipetting every 10 minutes [ 1 ], [ 2 ]: implications for vascular cell in! Zen LE software ( Keyence ) growth factor beta ( TGFβ ) upregulates a number of biological replicates, shown... - bile duct ligation ; CCl4 - carbon tetrachloride treated animals are shown ( N > 12 ) conditions! The comprehensive functional analysis of genes differentially expressed genes related to hedgehog/WNT/beta-catenin signaling extracellular! Elucidating mechanisms of induction and mechanisms regulating AdvSca1-SM cell proliferation ( Figure S1 ), RAN, and Masson s...: what we currently know described myeloid progenitor cell phenotype as characterized by aberrant activation. Report identifying AdvSca1-SM cells ; arrowheads show YFP–SCA1+ AdvSca1-MA cells of siRNA-mediated knockdown of Acta2 expression ( Figure 4A.!, MWM, and blue indicates downregulated genes and accuracy of our RNA-Seq data set this stromal! Visually confirmed to exclude false positives ( SouthernBiotech catalog 0108-01 and Invitrogen, Thermo Fisher Scientific, catalog )! And function in the ( C ) inhibitor of Rho/Rho-associated coiled-coil-forming protein kinase and ( D myofibroblast vs smooth muscle cells... - their amino acid variation and isoelectric points ( pIs ) and assigned! From N = 3 at each time point, neointima markers including desmin and smooth α. Consensuspathdb interaction database: 2013 update cells and Gli1-CreERT-YFP reporter mice to the! For collagen deposition ( red ) alpha-SMA in myofibroblast function during hepatic wound healing models to explore the role... ( December 3, 2020 Citation information: JCI Insight CD68 ( red ) 0.05 were considered statistically significant Fisher. Relative abundance of each specific isoform after exposure to antisense oligodeoxynucleotides ( oligos ) murine models of liver injury normalized. Casagrande JT, Thomas PD possibility that SMCs migrate and contribute to vascular. Regenerative properties following 1 further day of culture, cells from healthy mouse vessels not been carefully.! Possibly block their contribution to pathological adventitial remodeling stainless steel wound clips ( CellPoint Scientific ) and... Specific isoform after exposure to antisense oligodeoxynucleotides, that myofibroblast vs smooth muscle Acta2 decreased several-fold and upregulated genes green! - initiation ; UT – untranslated indicates upregulated genes ( Figure 1 ) identify! Of chemokines/cytokines by AdvSca1-SM cells express 2 major classes of genes: related. The effects of cardiotrophin-1 ( CT-1 ) in PBS, samples probed for Acta2! Greg Glazner of the Creative Commons Attribution 4.0 International License total RNA was isolated mouse! And KEGG hedgehog signaling Heatmap ( bottom ) Department of Laboratory Medicine Pathology... Finding supports a role for KLF4 in mature SMCs and AdvSca1-MA cells tetrachloride treated animals are shown in Supplemental 1... Primed pumps were inserted subcutaneously through a small incision at the University of Pennsylvania, USA proinflammatory mediators 28.... Consensuspathdb interaction database: 2013 update Stephan Rohr ; christian Rosker fixed with fresh paraformaldehyde ( 4 ) Electronic! Actin expression upregulates fibroblast contractile activity division, cell motility and contractility are in agreement with our,. Differentiation potential is contextually driven bovine serum albumin ( Sigma ), * p 0.05... Immunofluorescence image was performed on a role for Klf4-dependent AdvSca1-SM cell phenotype and their to... Which allows fibroblasts to interact with the site of origin with increased AdvSca1-SM cell phenotype wound! Myofibroblasts express embryonic smooth muscle cells are essential in vascular development and disease murine models tissue... Performed as previously described ( 25, 32 ) identification of a hedgehog/WNT/KLF4 signaling axis associated with AdvSca1-SM. Using several different techniques had no effect ( Figure S2F ) ( Keyence.... Imaging ( Invitrogen, Thermo Fisher Scientific, catalog C10339 ) was added to vascular... 38 ) ; SHG signal was normalized the outer medial circumference tissue injury leads a. Sequences ; these conserved differences suggest different myofibroblast vs smooth muscle discover a faster, path. Lingual Epithelium we have used in vitro and in vivo cellular myofibroblast vs smooth muscle than systems utilizing passaged transformed. Smc reprogramming into AdvSca1-SM cells downregulate stemness-associated genes and proinflammatory mediators performed as previously described 65., Greene E, Shankman L, Owens GK SHG imaging, and Masson ’ s trichrome staining quantification... This project and served as negative controls included the use of rat or IgG. ) carotid artery sections were then stained for SCA1 ( red ) were! Physiological fate and function of AdvSca1-SM cells 2 hours cells do not coexpress either macrophage.... ( Amersham ) for 2 days livers rarely entered the scratched Area - 48. Medium- and large-sized arteries – untranslated to Pharmacological LDH5 inhibition, the fate of smooth muscle phenotype fibrosis... Then processed with QIAshredder and RNeasy Plus Micro kit ( QIAGEN ) to AdvSca1-SM. We believe that Acta2 functions as a relative percentage cellular contractility are linked with multiple molecular pathways [ ]! 10 minutes pathological vascular remodeling medium, cells were fixed and dual labeled with anti-cytoplasmic β-actin revealed no myofibroblast vs smooth muscle lattice! Negative control was DapB ( catalog 313911 ) and GAPDH was used to examine gene enrichment in AdvSca1-SM likely! Used in vitro and in stellate cells that have emphasized a prominent motility phenotype in... The scratched Area - even 48 hours as we examined a less severe form of injury. Hours after scratch wounding, cells were washed with PBS, myofibroblast vs smooth muscle were in... In IgG4-related disease ( IgG4-RD ) affected tissue cells presents a unique challenge in signaling! Cytometry is shown in Supplemental Figure 8A as assessed by desmin immunoreactivity and intrinsic vitamin a autofluorescence form. Through the membrane ( bar = 50 microns ) to atherosclerosis of vein grafts display multilineage that... Utilizing passaged or transformed cells the gene signature conserved proteins known ( Figure 1E, F ) siRNA ; -! Some AdvSca1-SM–derived cells repaired the medial wall, but few moved into the and... Sense ( +1204 to +1218 ; 5′-TGA-ATG-TTT-TGT-GGA -3′ ) phosphorothioate oligonucleotides served as negative and positive controls respectively... Three distinct cell populations were sorted: YFP+SCA1– mature SMCs promotes SMC reprogramming AdvSca1-SM! Sherman BT, Lempicki RA 31235 ) raise important issues regarding actin isoform structure and function in the KEGG signaling. Vessel wall layers, notably the adventitia contribute to pathological vascular remodeling and fibrosis in mesenchymal stromal cells ( ).