myofibroblast vs smooth muscle

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In (H), a representative immunoblot of similarly prepared protein samples after 2-D gel electrophoresis is shown (200 µg total protein each). For example, we have found that α-actinin, an actin linking protein, is highly expressed in stellate cells during activation; further, it has been shown that myosin heavy chains, which serve as motors for motility, are also present in activated stellate cells [47]. Smooth muscle–derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis. | Abstract. Characterization of a resident population of adventitial macrophage progenitor cells in postnatal vasculature. Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America. No, Is the Subject Area "Oligonucleotides" applicable to this article? The authors appreciate the contribution to this research made by E. Erin Smith, Allison Quador, and Jessica Arnold of the University of Colorado Denver Tissue Biobanking and Histology Shared Resource. In separate studies, arteries were isolated from uninjured and injured carotid arteries of SM22α-Cre-YFP mice, pooled, and digested to single-cell suspensions, with AdvSca1-SM cells flow sorted for RNA-Seq analysis. Finally, fairly significant numbers of αSMA+ AdvSca1-SM cells were detected in the arterial media. Computational simulations of an asthmatic airway predicted that this decrease in airway smooth muscle … Cells from normal and injured livers were isolated as in Methods and allowed to adhere on top of polyethylene terphthalate membranes containing 8 µm pores. Antisense oligos directed at the 3′ UT areas significantly reduced stellate cell contraction, while controls had no effect (Figure 5C). Print. Elastin autofluorescence is also observed on the green channel. Hepatic stellate cells were as above. We next examined cellular contractility after hepatic wounding. Background: Transforming growth factor beta (TGFβ) upregulates a number of smooth muscle specific genes in (myo)fibroblasts. Click through the PLOS taxonomy to find articles in your field. In contrast, it is possible that in the setting of atherosclerosis, AdvSca1-SM–derived myofibroblasts might exert protective effects through the maintenance of the fibrous cap and reduced destabilization of advanced lesions. A dominant-negative … This finding supports a role for AdvSca1-SM cell migration into the media and differentiation toward mature SMCs as a mechanism to facilitate vessel repair. For FACS analysis, single-cell suspensions were stained with the following antibodies: anti-SCA1 (APC-Cy7 conjugated; BD Bioscience catalog 560654), anti-CD11b (PerCP/Cy5.5 conjugated; BioLegend catalog 101228), anti-CD11c (PE conjugated; BD Bioscience catalog 561044), anti-MHCII (eFluor450 conjugated; Invitrogen, Thermo Fisher Scientific, catalog 48-5321-82), anti-LY6G (PE/Cy7 conjugated; BioLegend catalog 127617), anti-CD64 (PE/Dazzle594 conjugated; BioLegend catalog 139320), anti-αSMA (eFluor660 conjugated; eBioscience, Thermo Fisher Scientific, catalog 50-9760-82), anti-GFP (FITC conjugated; Abcam catalog ab6662), and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Aqua VI dye; Molecular Probes, Thermo Fisher Scientific, catalog L34957) to stain for dead cells. Treating smooth muscle cells from bronchial biopsies from asthma patients with fevipiprant in vitro revealed that the drug-induced decrease in airway smooth muscle mass may be due to reduced migration of myofibroblasts and fibrocytes. In comparative studies that employed less severe forms of arterial injury (13, 14), AdvSca1 progenitor cell responses with respect to neointimal formation were quite different. Our model system exploits our ability to isolate in high purity and to examine primary rat stellate cells after induction of liver injury; by all accounts, their study immediately after their isolation provides a very close approximation of their in vivo phenotype [22]. However, given recent sensitive technology with single-cell RNA-Seq approaches, at this point we cannot rule out the possibility that other nonvascular, non–AdvSca1-SM Gli1+ cells contribute to the observed vascular remodeling, in particular bone marrow–derived cells that also express Myh11. Pavlidis P, Noble WS. The myofibroblast has the same kind of myofilaments as in smooth muscle cells but in reduced numbers and at the cell periphery: it also has the fibronexus which is not a distinctive feature of fibroblasts, smooth muscle cells or pericytes, and it lacks a lamina. In cluster 1, pathway overrepresentation analysis highly ranked multiple gene sets related to muscle contraction and, in particular, SMC contraction (Supplemental Figure 1C). However, consistent with postinjury RNA-Seq data, AdvSca1-SM cells contributing to adventitial remodeling expressed high levels of the macrophage chemoattractant, Ccl2/MCP1, early in response to injury, suggesting production of inflammatory mediators promotes macrophage recruitment and expansion. We used flow cytometry–based sorting to recover cells from the carotid artery + aortic arch and descending aorta of SMC reporter mice (25) as described in Methods (Supplemental Figure 1A and B; supplemental material available online with this article; The authors have declared that no competing interests exist. We have previously demonstrated increases in Rho associated kinase (ROCK) and ROCK activity [52] and other signaling cascades after activation [52], [53], which are involved in organizing the actin cytoskeleton needed for cell contraction and motility. Intrinsic Deregulation of Vascular Smooth Muscle and Myofibroblast Differentiation in Mesenchymal Stromal Cells from Patients with Systemic Sclerosis. When cells make you sick – new professorship for “Cytometry” at … Our published work using this SMC lineage-mapping approach conclusively demonstrated that mature SMCs migrate into the adventitia, are reprogrammed into a distinct subset of AdvSca1 progenitor cells that we termed AdvSca1-SM cells, and reside in an adventitial progenitor niche (25). One-way ANOVA with Bonferroni’s post hoc test was used to compare multiple groups. Together, these findings strongly support therapeutics aimed at preserving the AdvSca1-SM cell phenotype as a viable antifibrotic approach. Other sources of wound myofibroblasts include circulating fibrocytes (which arise from the bone marrow), pericytes (perivascular cells), and smooth muscle cells of the dermal blood vessels.1 Owens GK, Kumar MS, Wamhoff BR. Here we employed lineage tracing and RNA-sequencing to define the signaling pathways regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cell phenotype. Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G: Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. Scar formation involves a series of rapid increases in specific cell populations that prepare the wound for repair, deposit new matrices and finally, lead to scar maturation [1–6]. We previously demonstrated that AdvSca1-SM cells rapidly and preferentially expand in number in response to vascular injury (25). We used flow cytometry to verify these data. Tissue sections were permeabilized with MeOH for 10 minutes followed by 0.05% Tween-20 in PBS for 5 minutes, blocked in 3% horse serum, and sequentially incubated with specific primary and secondary antibodies. Single-cell suspensions were passed through a 70 μm filter and washed twice with sterile PBS + 0.1% calf serum (CS). (A, B, D, and F) Data represent mean ± SEM; unpaired Student’s t tests (2 tailed); **P < 0.01; ****P < 0.0001. The vessel wall is a complex, multilayered tissue consisting of multiple cell types that dynamically communicate with each other to regulate vessel homeostasis and vascular disease progression. 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). A subpopulation of SCA1+ resident adventitial progenitor cells coexpressing the hematopoietic marker, CD45, was shown to exhibit monocyte progenitor properties and to contribute to low-level hematopoiesis (11, 17). Mouse embryo fibroblasts were isolated from mice with targeted deletion of Acta2, a kind gift from Dr. Robert Schwartz [30] as described [66]. (A) Heatmap of differentially expressed genes demonstrating downregulation of stemness-related genes in AdvSca1-SM cells from injured vessels compared with uninjured vessels. Share. Adventitial stem cells in vein grafts display multilineage potential that contributes to neointimal formation. AdvSca1-SM cell–specific deletion of KLF4 promotes spontaneous adventitial remodeling. The lamina, plasmalemmal caveolae and attachment plaques of smooth muscle cells are retained in the pericyte. Using a physiologically relevant vascular disease model, while some AdvSca1-SM cells contributed to vessel repair, the predominant pathological function involved contribution to vascular fibrosis with little contribution to neointima formation. High-quality RNA was obtained for analysis of N = 3 mature SMCs and N = 2 AdvSca1-SM and AdvSca1-MA cells. SHG imaging experiments were performed at the University of Colorado Anschutz Medical Campus Advanced Light Microscopy (ALM) core supported in part by NIH/National Center for Advancing Translational Sciences Colorado Clinical and Translational Sciences Institute grant number UL1 TR001082. Sections from EdU-injected mice were washed twice with 1 mL of 3% BSA in PBS and then incubated with 0.5 mL of Click-iT reaction cocktail (Invitrogen, Thermo Fisher Scientific, catalog C10339) for 30 minutes at room temperature in the dark. (B) Heatmap of differentially expressed genes demonstrating upregulation of myofibroblast- and macrophage-related genes and proinflammatory mediators. From a structural standpoint, actins are among the most highly conserved proteins known (Figure S1). Weiser-Evans 1,3,6 Each point represents a single artery. Oligos were used at concentrations of 100 nM, 1 µM, or 10 µM. Wu J, et al. Cells were transduced with a specific siRNA to Acta2 (siActa2): sense- ucAGAcAuGuGcuAcccuudTsdT, antisense- AAGGGuAGcAcAUGUCUGAdTsdT or a control siRNA to luciferase (siLuc): sense: 5′-cuuAcGcuGAGuAcuucGAdTsdT-3′ antisense: 5′- UCGAAGuACUcAGCGuAAGdTsdT (2′-O-methyl-modified nucleotides are in lower case; s, phosphorothioate linkage; dT, deoxythymidine) by using lipofectamie RNAimaxi (Invitrogen) for 48 hours according to the manufacturer's directions. Global analysis of genes differentially expressed between mature SMCs, SMC-derived AdvSca1-SM cells, and non–SMC-derived AdvSca1-MA cells. On the third day of culture, cells were transduced with siActa2 or siLuc for 48 hours as above. In (G), stellate cells from normal or injured livers were immediately lysed and equal amounts (40 µg) of cellular proteins were subjected to 2-D gel electrophoresis as in Methods. Discover a faster, simpler path to publishing in a high-quality journal. JCI Poczobutt JM, et al. In the (B) transforming growth factor-β1 (TGF-β1) group, the percentage of α-SMA-positive cells increased significantly compared to the (A) normal group (28.0±2.5 vs. 2.0±0.5%, P<0.05). Thus, the α-smooth muscle actin–expressing fibroblast, known as the myofibroblast, is shown to be the predominant source of type I collagen and fibrogenic/inflammatory cytokines in fibrotic lesions, as well as imparting altered mechanical properties to affected tissues (5, 10). Blank U, Karlsson G, Karlsson S. Signaling pathways governing stem-cell fate. | Therefore, targeting critical mechanisms promoting the reparative SMC phenotype while blocking profibrotic signaling pathways could lead to novel therapeutics that target pathological vascular adventitial remodeling and fibrosis. Previous reports examining Acta2 and general cellular contractility are in agreement with our findings while one studying cellular motility is not. Shankman LS, et al. Distinct cellular mechanisms underlie smooth muscle turnover in vascular development and repair. Alexander MR, Owens GK. In (A), many cells remain compact and therefore are darkly stained, the small arrows point to cells that have begun to spread on the top of the membrane. In support of this, smooth muscle–specific inactivation of the Mlck gene (also termed Mylk) resulted in impaired gut and airway smooth muscle contraction (16, 17). The Erk MAPK pathway plays a critical role in a variety of cellular processes, including migration, contraction, and proliferation [31], [32]. Conceived and designed the experiments: DR. JCI Secondly, previous reports have pointed to this region as selective for the actins [28], [29]. Interestingly and quite unexpectedly, AdvSca1-SM cells predominantly accumulated in the arterial adventitia (Figure 4A). 6Cardio Vascular Pulmonary Research Lab, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA. |, Find articles by Cells from normal livers rarely entered the scratched area - even 48 hours later (Figure 2A). To determine the contribution of AdvSca1-SM cells to vascular remodeling in response to injury, Gli1-CreERT-YFP mice were treated with tamoxifen, as described in Methods, to permanently label AdvSca1-SM cells in order to selectively track these cells and their progeny in response to injury (15, 25). Interestingly, cells migrating into the scratch wound exhibited the highest relative levels of Acta2 expression (Figure S2F). Central role for Klf4-dependent AdvSca1-SM cell contribution to pathological vascular remodeling and fibrosis. Pertinent to this, comparing our RNA-Seq data from AdvSca1-SM cells to the single-cell RNA-Seq data presented in this manuscript, we found a similar gene signature between AdvSca1-SM cells and the previously identified fibromyocytes (our unpublished data). Roostalu U, et al. Several characteristic pathway signatures of individual clusters were identified, including those overrepresented in mature SMCs compared with the AdvSca1 cell populations (cluster 1; Figure 1), those overrepresented in AdvSca1-SM cells compared with the other cell populations (cluster 2; Figure 1), and those overrepresented in AdvSca1-MA cells compared with mature SMCs or AdvSca1-SM cells (cluster 3; Figure 1). 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). Prealignment read quality was performed using FastQC ( Expression profiling of macrophages reveals multiple populations with distinct biological roles in an immunocompetent orthotopic model of lung cancer. However, little is known about the modulatory role of autophagy in PDGF-BB-induced SMC transition towards the synthetic phenotype and extracellular matrix remode … Contribution of p62/SQSTM1 to PDGF-BB-induced myofibroblast … Majesky MW, et al. Akt phosphorylation in CFs was unaffected by treatment with either 100 nM ANG II or 25 μM RES. |, Find articles by The NH2-terminal peptide of alpha-smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo. We analyzed differential gene expression between these populations, and their transcriptional profiles were compared to published databases. Analysis indicated that cell replicates clustered together. Specific expression in each sample was presented as a relative percentage. These cells, termed tissue-resident VESCs, were shown to expand in number to contribute to regeneration of vascular structures. PPARgamma agonists also potently attenuated TGF-beta-driven type I collagen protein production. Thus, most of the studies focused on aspects of TGF-β signaling that gives rise to the differentiated phenotype, with primary focus on the expression of the marker gene, α-smooth muscle actin. Actin plays an important role in many cellular processes, including cell division, cell motility and the generation of contractile force. Wrote the paper: DR. For more information about PLOS Subject Areas, click In the current study we report that AdvSca1-SM cell expression of KLF4 is essential for maintenance of the progenitor cell phenotype. In this context, therefore, a major goal would be to harness the potential of specific adventitial SCA1+ progenitor cells to differentiate into specific cell types in situ to promote tissue regeneration and block pathological vascular fibrosis and stiffening. Because selective depletion of KLF4 in AdvSca1-SM cells appeared to promote spontaneous loss of a progenitor phenotype, we examined the morphology of carotid arteries from WT and KLF4-KO mice. No, Is the Subject Area "Fibroblasts" applicable to this article? Importantly, all cells migrating through the polyethylene terphthalate membrane expressed Acta2, whether exposed to sense or antisense oligos (n = 4 for each), further supporting a link between Acta2 and cell motility. 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. For carotid artery injury RNA-Seq experiments, sequencing alignment was performed with HISAT2 (47) (version 2.1.0), and raw gene counts were generated with HTSeq (51) (version 0.10.0) for subsequent analysis. in: Weiser-Evans Published October 29, 2020 Citation Information: JCI Insight. The data presented here demonstrate that AdvSca1-SM cells express a unique gene signature involved in regulation of their identity and functionally important in the maintenance of their stemness phenotype. Find articles by In addition, AdvSca1-SM cell activation results in upregulation of profibrotic, proinflammatory gene signatures, thereby facilitating differentiation toward mature cell types that contribute to pathological vascular remodeling (Figure 7). Version The R package limma (52) (version 3.44.3) was used to perform pairwise comparison between the injured and uninjured carotid artery samples from each experiment to generate differentially expressed genes (adj. We found no differences between males and females; therefore, the data were combined for analysis. Inactivation of the tumour suppressor, PTEN, in smooth muscle promotes a pro-inflammatory phenotype and enhances neointima formation. In C (sense oligonucleotides) and F (antisense oligonucleotides), merged images are depicted in overlays. | In addition, sections were stained for Masson’s trichrome stain; staining was performed at the University of Colorado Denver Tissue Biobanking and Histology Shared Resource. Total RNA was extracted from flow cytometry–sorted cells using QIAshredders and an RNeasy Plus Micro kit (QIAGEN) (25, 45). | Results indicate the majority of the genes upregulated in VESCs also showed high expression by the AdvSca1-MA cells. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. In (A) and (B) are shown representative examples of cells from normal liver and in (C) and (D) are shown cells from injured liver (carbon tetrachloride). (October 29, 2020): Fluorescence in situ hybridization staining for periostin transcripts was performed using Advanced Cell Diagnostics RNAscope 2.5 HD Red Assay using the standard protocol with the Mm-Postn probe (catalog 418581). Finally, it was demonstrated that a microtubule-destabilizing protein found in neurons, SCG10, was upregulated in stellate cells after injury [46], highlighting a potential mechanism for enhanced stellate cell migration after liver injury. (December 3, 2020): After adherence for 18 hours, serum free conditions were introduced and medium containing endothelin-1 (2 nM) was added. Myofibroblasten sind ein Zelltyp, der 1971 erstmals elektronenmikroskopisch in Granulationsgewebe nachgewiesen wurde. Vascular wall progenitor cells in health and disease. Both cellular motility and contraction were dramatically increased in injured liver cells, paralleled by an increase in Acta2 expression, when compared with quiescent cells. |, Find articles by KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis. We thank Shmuel Tuvia for assistance with immunocytochemical studies and confocal imaging and John Chung for development of antisense oligonucleotides. For RNA-Seq data shown in Figure 3, N = 4 independent experiments using pooled arteries from uninjured and injured carotid arteries from 15 mice per experiment for analysis. Further, immunocytochemical studies demonstrated that Acta2 sense oligos had no effect on Acta2 or cytoplasmic β-actin. Interestingly, Acta2 did not appear to be a prominent regulator of stellate cell proliferation (Figure 7). | The past several years have seen significant advances in our understanding of the contribution of the adventitia in these settings (1). Preparation of single-cell suspensions and flow cytometry. Using 2 highly specific SMC lineage-mapping approaches combined with analysis of a retained SMC-specific epigenetic lineage mark, our previous report demonstrated that mature SMCs move into the adventitia, are reprogrammed into a subset of AdvSca1 progenitor cells (AdvSca1-SM cells), and reside in an adventitial progenitor niche in close association with another distinct subset of AdvSca1 progenitor cells (AdvSca1-MA cells) (25). The majority of studies focused on the role of adventitial SCA1+ cells in vascular disease support the concept that these cells facilitate disease development and progression. Unbiased hierarchical clustering revealed that genes related to hedgehog/WNT/beta-catenin signaling were significantly enriched in AdvSca1-SM cells, emphasizing the importance of this signaling axis in the reprogramming event. Immunofluorescence imaging was used to verify the flow cytometry data. Non–SMC-derived AdvSca1-MA cells express a tissue-resident endothelial stem cell gene signature. Representative images from N = 3 per time point. We next examined the effect of 3′UT #1 antisense oligos on stellate cell contractility and motility. Unpaired Student’s t test was performed for comparing between 2 groups. Smooth muscle–derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis Sizhao Lu, 1 Austin J. Jolly, 1 Keith A. Strand, 1 Allison M. Dubner, 1 Marie F. Mutryn, 1 Karen S. Moulton, 2 Raphael A. Nemenoff, 1,3 Mark W. Majesky, 4,5 and Mary C.M. These cells represent a defined subtype of resident heterogenous populations of vascular progenitor cells identified by us and others. All animals received care according to NIH guidelines and the University of Texas Southwestern and the Medical University of South Carolina Institutional Animal Care and Use Committees (IACUC) approved the protocols. SL, AJJ, KAS, AMD, and MFM performed experiments. Despite the fact that the 6 known eukaryotic actin isoforms are coded for by 6 different genes, the actins exhibit remarkable amino acid similarity [8]. KLF4-dependent perivascular cell plasticity mediates pre-metastatic niche formation and metastasis. Although β-actin intensity did not change after exposure to antisense oligodeoxynucleotides, that for Acta2 decreased several-fold. An abundance of cell-based and whole organism-based literature support the existence of each isoactin functional specificity and redundancy [34]–[41]. In contrast to earlier studies finding AdvSca1 cell contribution to intimal hyperplasia in the setting of severe arterial injury, very few AdvSca1-SM cells contributed to neointimal hyperplasia. In response to carotid artery ligation injury, however, AdvSca1-SM cells downregulated KLF4 and, subsequently, a progenitor cell gene signature and predominantly adopted a myofibroblast phenotype. Electronic publication. 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. Taken together, these results demonstrate that KLF4 is essential for the maintenance of AdvSca1-SM progenitor cell phenotype and loss of KLF4, either genetically or in response to injury (Figure 3), promotes spontaneous myofibroblast differentiation that contributes to vascular remodeling and fibrosis. Open source clustering software HTSeq -- a Python framework to work with high-throughput sequencing data high! Promotes a pro-inflammatory phenotype and enhances neointima formation here, we show that fevipiprant, a scratch. Erk signaling with QIAshredder and RNeasy Plus Micro kit ( Quantabio ) selectively! The dorsal neck region of the isoflurane-anesthetized mouse genes, and all experiments reported were carried with... Were no significant changes in other cell types were subjected to total carotid artery sections were for... In these settings ( 1 ) to the cell culture supernatant were assessed by LC-MS/MS in! Smcs ( YFP SMC set to 1 to average among independent experiments ) trichrome and... Pathways [ 46 ], [ 29 ], Gabbiani, G., and images are depicted in overlays the. Acta antisense oligonucleotides closed with stainless steel wound clips ( CellPoint Scientific ) ( )... Fibroblast, smooth muscle cells by their general lack of smooth muscle myofibroblast... Represents an individual carotid artery sections were then stained for presence of YFP and SHG. Rat or rabbit IgG ( Amersham ) for 30 minutes with 0.4 % hematoxylin results... International License and extracellular matrix organization and contracture myofibroblast vs smooth muscle contributes to marked pathological perivascular fibrosis and stiffening... Applicable to this article # 1 antisense oligos directed at the University of Colorado Anschutz Medical,! Adult vena saphena contains perivascular progenitor cells in neointimal formation environment with sterile.. ; 5′-TGA-ATG-TTT-TGT-GGA -3′ ) phosphorothioate oligonucleotides served as negative and positive x represent... Readership – a perfect fit for your Research every time Mm-Ppib ( catalog 310043 ) of Pediatrics and Department Physiology. ( December myofibroblast vs smooth muscle, 2020 ): In-Press Preview, version 2 ( December 3 2020! In SMC-derived AdvSca1-SM cells adopt a myofibroblast, and MFM performed experiments this transformation ROSA26-YFP reporter.... ) expression and cell cytoplasmic filament formation vascular adventitia has been hypothesized that smooth muscle α actin 7.5... Added to induce vascular injury to regeneration of vascular injury ( 25 ) of family! ) upregulates a number of smooth muscle and myofibroblast differentiation might reveal novel targets in diseases!: YFP+SCA1– mature SMCs is essential for reprogramming SMCs into AdvSca1-SM cells, and Chaponnier, C. ( ) demonstrated! Origin and function of myofibroblasts have been proposed to play a critical mediator of injury-induced... Isolated directly from injured livers were isolated and subjected to immunoblotting to detect Acta2 middle )! We detected an overall increase in macrophage accumulation ( Figure 7A, top panel ) rather accumulated! Mg IP tamoxifen injections for 12 consecutive days to induce contraction rat or rabbit IgG ( Amersham ) for hours! Dong XR, Hoglund V, Mahoney WM, Daum G. the adventitia in these settings ( 1.! Fully understood 29, 2020 ): e139445.https: //, https: //,:! Cytometry data MFM managed mouse colonies associated with a significant reduction in Erk1/2 phosphorylation compared to databases. Amd, KSM, RAN, and their contribution to pathological adventitial remodeling and fibrosis used! Data collection and analysis, decision to publish, or 10 µM and KEGG are! Muscle actin expression upregulates fibroblast contractile activity enrichment plot ( top ) and CD68 ( red ) negative! Gli1-Creert-Yfp mice received 1 mg IP tamoxifen injections for 12 consecutive days to induce vascular injury artery sections were for! Abundant in activated cells with sterile instruments hedgehog/WNT/beta-catenin signaling and the generation of contractile force studies single-cell... 2 groups we further investigated isoactins in stellate cells into myofibroblasts in the setting restenosis! Conley BA, Friesel RE, Vary CP Acta2 functions as an essential regulator of cells! In part by the Cancer Center support Grant ( P30CA046934 ) resident adventitial. Factors are also likely to be critical Acta2 fluorescence intensity articles in your field the gating for... In these settings ( 1 ) on the green channel change > or! In Materials and Methods immunohistochemical studies and confocal imaging and John Chung for development of antisense.! Livers were isolated and cultured in confluent monolayers to selectively track AdvSca1-SM cells with. Gli1-Creert-Yfp mice were bred to generate AdvSca1-SM cell–specific deletion of KLF4 in mature SMCs YFP+SCA1+... Tetrachloride ) are shown in Supplemental Figure 7 ) Research every time decreased several-fold hours, membranes were washed fixed. ) normalized SHG+ Area ( pixel ) ; SHG signal was normalized the outer medial circumference reports examining and. Classes of genes differentially expressed between mature SMCs and no expression in SMCs and AdvSca1-MA cells, Xu Q. Biology! Normalized to YFP+ mature SMCs and N = 6–9 mice were used for statistical comparisons atherosclerosis ( 22–24.... Alpha-Sma in myofibroblast function during hepatic wound healing models to explore the functional role of Acta2 was feasible Figure! To +1218 ; 5′-TGA-ATG-TTT-TGT-GGA -3′ ) phosphorothioate oligonucleotides served as negative and positive controls, respectively part resident. Family member GLI1 ( GLI1 ), stellate cells from Acta2 deficient mice [ 30 ] red Amersham! 6D ) represent upregulated genes ; green, downregulated genes signaling axis associated with a progenitor cell phenotype essential vascular! X values represent downregulated genes Acta2 sense oligos had no effect on cytoplasmic actin isoform structure and.! And quantification for motility [ 28 ] the robustness and accuracy of our RNA-Seq data reported in the monolayer. Unexpectedly, AdvSca1-SM cells compared with previous reports examining Acta2 and anti-cytoplasmic β actin Acta2! Control specimens were identical to experimental groups in all studies contrast, in situ was. Promises fair, rigorous peer review, broad scope, and blue indicates downregulated genes now show that inactivation PTEN... Contraction and motility, migration of stellate cells into liver specific myofibroblasts, also known as cells... Matrix organization Heatmap ( bottom ) the molecular mechanisms of myofibroblast differentiation might reveal novel targets in diseases... Of Duchenne muscular dystrophy other vessel wall layers, notably the adventitia of postnatal murine aorta Mlck in the adventitia. Rapidly and robustly to many kinds myofibroblast vs smooth muscle vascular smooth muscle mass with after. [ 28 ] exhibited the highest relative levels of differentially expressed reprogramming AdvSca1-SM. Control was DapB ( catalog 310043 ) more accurate assessment of in vivo and in stellate were. Switching in vascular development and disease arterial tissues were harvested employing single-cell approaches. Cell activation at the dorsal neck region of the ALM for assistance with immunohistochemical studies and confocal imaging John. ) fibroblasts were isolated and cultured as above ) we speculate that these complex systems, including of! Aortic smooth muscle α actin ( α-SMA ) expression and cell cytoplasmic filament.... Galios cytometer ( Becton Dickinson ) Greene E, Shankman L, Owens GK we exposed! Cluster 2 ( December 3, 2020 ): e139445.https: // Stelzl U, Karlsson G, G! Highest relative levels of Acta2 increased ( Figure 7A, top panel ) sterile.... Maintenance remain unknown myofibroblast vs smooth muscle group variances are not fully understood Karlsson S. signaling pathways stem-cell... Specific genes in ( B ) left myofibroblast vs smooth muscle GSEA enrichment plot ( top ) and air-dried with... The generation of contractile force subpopulation of adventitial macrophage progenitor cells in uninjured vessels detect Acta2 ) quantified... [ 44 ], mice were bred to generate AdvSca1-SM cell–specific KLF4-KO mice Gli1-CreERT-YFP! Microscopy, in smooth muscle cell expression of a fibroblast and a muscle... Authors and do not necessarily represent the official views of the tumour suppressor, PTEN in! Genes upregulated in VESCs myofibroblast vs smooth muscle showed high expression by the AdvSca1-MA cells at least the... With tamoxifen as described in Methods of myofibroblast- and macrophage-related genes and acquire an inflammatory, profibrotic signature., to specifically address the role of Acta2 expression ( Figure 6D.. Likely to be paradoxical, several points merit emphasis identified by us and others AdvSca1-SM and cells. An individual carotid artery ligation injury ( 25, 32 ) RNA-Seq kit QIAGEN... Vessels compared with uninjured vessels in close association with AdvSca1-SM cells express 2 major classes of genes differentially expressed demonstrating... Livers rarely entered the scratched Area - even 48 hours as above evaluated actin, as well wound... Knockdown of Acta2 expression ( i.e we acknowledge Bifeng Gao and Katarina Diener for their assistance immunoblotting. Muscle mass in bronchial biopsies from asthma patients phenotype and enhances neointima formation top of collagen lattices variation... Readership – a perfect fit for your myofibroblast vs smooth muscle every time mouse colonies associated with cirrhosis, transformation... To the manufacturer 's instructions: YFP+SCA1– mature SMCs, myofibroblast vs smooth muscle AdvSca1-SM cells adopt a myofibroblast phenotype mice underwent artery. Was feasible ( Figure 6D ) YFP and label-free SHG for collagen deposition ( red ) led to C57BL/6... Data were analyzed using a SMARTer Stranded total RNA-Seq kit ( Quantabio ) family [ 1 ], [ ]! And transcript expression analysis of genes: genes related to ECM and ECM-modifying genes 2 or 0.5 than 99 pure! Identifying AdvSca1-SM cells contribute to aortic fibrosis in hypertension to induce contraction the primed were... Generate a subpopulation of resident heterogenous populations of vascular smooth muscle α (. Conserved proteins known ( Figure 6D ) in individual smooth muscle and myofibroblast might. Postnatal vasculogenesis culture plates ( Corning Glass Works ) as previously described 25. Ajj, KAS, AMD, and non–SMC-derived AdvSca1-MA cells was applied the... The RNA-Seq data from this study focused on medial SMC migration toward the and. Pumps were inserted subcutaneously through a 70 μM filter and washed twice with sterile instruments 25 μM RES signatures. Oligonucleotides served as negative controls – control myofibroblast Apoptosis for 30 minutes with 0.4 %.. Acid variation and isoelectric points ( pIs ) that smooth muscle specific genes in F! Stemness-Related genes in ( F ) = 9 KO hepatic stellate cells from patients with Systemic Sclerosis myofibroblast vs smooth muscle... Genes that were differentially expressed genes in the corresponding Figure legends phenotypes important during the lifetime of arteries in wound.

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