Myofibroblasts may arise from the transdifferentiation of fibroblasts and smooth muscle cells. In cluster 1, pathway overrepresentation analysis highly ranked multiple gene sets related to muscle contraction and, in particular, SMC contraction (Supplemental Figure 1C). Google Scholar Studies on these phenotypic transitions have mostly focused on medial SMC migration toward the intima and their contribution to intimal hyperplasia and inflammation. 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). 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. Again, cells early after isolation were studied, prior to culture-induced changes or potential artifact, so as to allow a direct analysis of their in vivo phenotype. in: | 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). Each experiment utilized cells from a different animal. Smooth muscle α actin (Acta2) expression is largely restricted to smooth muscle cells, pericytes and specialized fibroblasts, known as myofibroblasts. In contrast to previous work (22, 23, 36), a recent intriguing paper employing a similar Myh11-CreERT SMC fate-mapping system and single-cell RNA-Seq approaches in the setting of atherosclerosis reported that mature SMCs transition to “fibromyocytes” rather than macrophages to contribute to fibrous cap formation and lesion stability (42). Interestingly, this was associated with an overall increase in the number of AdvSca1-SM cells in KLF4-KO mice compared with WT mice (Figure 6B and Supplemental Figure 8C). After washing with PBS, samples were incubated with streptavidin-linked Texas Red (Amersham) for 30 minutes, washed again and mounted. (B) Uninjured and 3- and 7-day postinjury carotid artery sections were immunofluorescently stained for YFP (green) to identify AdvSca1-SM cells. As shown in Figure 3A, genes associated with hedgehog/WNT signaling (e.g., Gli1, Gli2, Ptch1, Wnt2b, and Wnt5a), stemness-associated genes (e.g., Klf4, Ly6a/SCA1, Cd34), and several epigenetic regulators (e.g., Tet1, Hdac7, Prdm6) were found downregulated in AdvSca1-SM cells in response to injury. Adventitial stem cells in vein grafts display multilineage potential that contributes to neointimal formation. Others have shown that SMC-specific depletion of KLF4 attenuates experimental atherosclerosis lesion formation and abdominal aortic aneurysm formation (23, 44). Together, these findings strongly support therapeutics aimed at preserving the AdvSca1-SM cell phenotype as a viable antifibrotic approach. Hu Y, et al. AdvSca1-SM cells exhibit a multipotent phenotype marked by their ability to differentiate into multiple cell types in response to specific environmental cues, including SMCs, endothelial cells, macrophages, myofibroblasts, and adipocytes. Rather, accumulated macrophages, especially in the adventitia, appeared to cluster in close association with AdvSca1-SM cells. Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels. Subramanian A, et al. KLF4 is a member of the Kruppel-like family of transcriptional regulators. PLoS ONE 8(10): In addition, compared with uninjured control arteries and consistent with our RNA-Seq data (Figure 3), following injury AdvSca1-SM cells gradually lost expression of SCA1, suggesting activation and differentiation of these cells into another cell fate. Multiple positive-regulatory elements were identified as essential mediators of basal SmαA … Because Bst1 was also found to be upregulated in AdvSca1-MA cells compared with AdvSca1-SM cells and mature SMCs, to determine the similarity of AdvSca1-MA cells to these previously described VESCs, we compared the 5265 differentially expressed genes generated from our data set with the top 500 upregulated genes in the VESC population from this study and generated a heatmap with the overlapping genes (Supplemental Figure 3A). Guided by our RNA-Seq findings demonstrating AdvSca1-SM cells uniquely expressed high levels of the transcription factor, Gli1, we generated an AdvSca1-SM cell fate-mapping system. Identification of a monocyte-predisposed hierarchy of hematopoietic progenitor cells in the adventitia of postnatal murine aorta. No, Is the Subject Area "Immunoblotting" applicable to this article? Classically, the presence of alpha smooth muscle actin (aSMA) has been considered a marker of myofibroblast … Myofibroblast differentiation is commonly induced by treatment of fibroblasts or other susceptible precursor cells with TGF-β. Huang da W, Sherman BT, Lempicki RA. Citation: Rockey DC, Weymouth N, Shi Z (2013) Smooth Muscle α Actin (Acta2) and Myofibroblast Function during Hepatic Wound Healing. Top 20 pathways identified through full pathway analyses conducted through Gene Ontology and KEGG databases are shown in Supplemental Tables 2–5. Myofibroblasten sind ein Zelltyp, der 1971 erstmals elektronenmikroskopisch in Granulationsgewebe nachgewiesen wurde. Majesky, M. | KLF4 regulates abdominal aortic aneurysm morphology and deletion attenuates aneurysm formation. However, most studies that have focused on these cells examined heterogenous populations of AdvSca1 cells rather than defining distinct functions of phenotypically unique subpopulations. , In this study, we show that pulmonary myofibroblasts express three of the eight … The gating strategy for sorting is shown in Supplemental Figure 1. Owens GK, Kumar MS, Wamhoff BR. Salmon M, et al. Fibroblasts produce the extrac… (E) Percentage of YFP+αSMA+ cells per total YFP+ cells. The differentiation of fibroblasts into myofibroblasts is featured by the increase of α-smooth muscle actin (α-SMA) expression and cell cytoplasmic filament formation. To test whether AdvSca1-SM cells contribute to myofibroblast or macrophage accumulation in response to injury, uninjured and injured carotid arteries were analyzed for expression of cell-specific markers. We have previously demonstrated increases in Rho associated kinase (ROCK) and ROCK activity  and other signaling cascades after activation , , which are involved in organizing the actin cytoskeleton needed for cell contraction and motility. Kramann R, et al. (B and C) Arterial sections were immunofluorescently stained for YFP (green) and CD68 (B; red) or MAC2 (C; red). The stellate cell to myofibroblast transformation process, also known as “activation” - in which Acta2 is an integral component - appears to be analogous to that occurring in fibroblasts after injury and wound healing in other pathological settings , –. Analysis indicated that cell replicates clustered together. Coronary adventitial cells are linked to perivascular cardiac fibrosis via TGFβ1 signaling in the mdx mouse model of Duchenne muscular dystrophy. 29 The population of myofibroblasts is thought to come from the blood, as well as an epithelial to … SL and MCMWE designed the studies. 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). To quantitate SCA1 expression and total YFP+ AdvSca1-SM–derived cells, arteries from Gli1-CreERT-YFP WT and Gli1-CreERT-YFP Klf4fl/fl KO mice were individually digested to single-cell suspensions, labeled with APC-conjugated anti-SCA1 antibodies, and analyzed for endogenous YFP and SCA1-APC expression. Google Scholar Google Scholar GSEA tool was used to examine gene enrichment in AdvSca1-SM cells compared with mature SMCs and AdvSca1-MA cells. The incision was closed with stainless steel wound clips (CellPoint Scientific). Preparation of single-cell suspensions and flow cytometry. 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. Interestingly and quite unexpectedly, AdvSca1-SM cells predominantly accumulated in the arterial adventitia (Figure 4A). Biol. https://doi.org/10.1371/journal.pone.0077166.g006. Our previous work demonstrated that induction of KLF4 in mature SMCs is essential for reprogramming SMCs into AdvSca1-SM cells (25). We now report that MyoD converts normal dermal fibroblasts, chondroblasts, gizzard smooth muscle, and pigmented retinal epithelial cells into elongated postmitotic mononucleated striated myoblasts. The sarcomeric localization of antibodies to desmin, alpha-actinin, titin, troponin-I, alpha-actin, myosin heavy chain, and myomesin in these converted myoblasts are … Global analysis of genes differentially expressed between mature SMCs, SMC-derived AdvSca1-SM cells, and non–SMC-derived AdvSca1-MA cells. here. Li G, et al. Upon resolution of inflammation, this myofibroblast-mediated stromal reaction typically disappears. (A) Left: GSEA enrichment plot (top) and KEGG hedgehog signaling heatmap (bottom). Both natural (15d-PGJ2) and synthetic (ciglitazone and rosiglitazone) PPARgamma agonists inhibited TGF-beta-driven myofibroblast differentiation, as determined by alpha-smooth muscle actin-specific immunocytochemistry and Western blot analysis. RNA-Seq reads were obtained using Illumina HiSeq analysis pipeline, as previously described (46). To analyze collagen deposition, arterial sections were immunofluorescently stained for YFP to identify AdvSca1-SM cells and sections imaged for YFP expression and label-free SHG using a laser-scanning confocal microscope (LSM 780). Bone marrow-derived mesenchymal stromal cells (MSCs) from SSc patients may harbor disease-specific abnormalities. Phosphatidylinositol-3-kinase signaling mediates vascular smooth muscle cell expression of periostin in vivo and in vitro. | doi: 10.1371/journal.pone.0153101. In (F), the number of cells per high-powered field entering the wounded area of the monolayer were counted and quantitated as in Methods (n = 6, *p<0.01 vs. cells exposed to sense oligonucleotides). Of note, cells migrating into the scratched areas appeared to exhibit more intense Acta2 labeling than cytoplasmic β-actin expression (Figure 4F); this was verified by demonstrating that quantitative fluorescence intensity in cells migrating into scratch-wounded areas was greater for Acta2 than for cytoplasmic β-actin. All surgeries were performed in a clean environment with sterile instruments. We also thank Alfica Sehgal for providing Acta2 and control (luciferase) siRNA. Distinct cellular mechanisms underlie smooth muscle turnover in vascular development and repair. |, Find articles by This process involved downregulation of Klf4 activity and induction of a profibrotic myofibroblast phenotype. Importantly, retention of YFP expression by AdvSca1-SM cells permitted faithful tracking and quantification in response to injury even though these cells lost all characteristics of AdvSca1-SM cells (e.g., loss of SCA1 expression). Lactate and pyruvate levels in the cell culture supernatant were assessed by LC-MS/MS. In another study, it was likewise shown that activated stellate cells were motile , and additionally that inhibition of the myosin II ATPase with blebbistatin, stimulated stellate cell migration. Total RNA was isolated from flow-isolated cell populations by first digesting in RLT lysis buffer (QIAGEN) (25, 45). In addition to pharmacological LDH5 inhibition, the effect of siRNA-mediated knockdown of LDHA … Atheroprotective roles of smooth muscle cell phenotypic modulation and the TCF21 disease gene as revealed by single-cell analysis. Kim D, Langmead B, Salzberg SL. Single-cell suspensions were passed through a 70 μm filter and washed twice with sterile PBS + 0.1% calf serum (CS). In C (sense oligonucleotides) and F (antisense oligonucleotides), merged images are depicted in overlays. Future studies employing single-cell RNA-Seq approaches will be useful to fully characterize the heterogeneity of this subtype of AdvSca1 cells. In (D), the area in the scratch remaining unoccupied by cells was quantitated (in each experiment, 10 random fields were assessed; the area remaining free of cells was measured by image analysis as in Methods, single data points were created for each experiment and were used to generate quantitative data; n = 6 for each model of injury). Elastin autofluorescence is also observed on the green channel. Skip main navigation. In particular, cell motility and contractility appear to be stellate cell phenotypes important during the wounding response. (H) CA and aortic (AO) sections from WT or AdvSca1-SM cell–specific KLF4-KO mice were immunofluorescently stained for YFP (green) to identify AdvSca1-SM cells. 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. High-quality RNA was obtained for analysis of N = 3 mature SMCs and N = 2 AdvSca1-SM and AdvSca1-MA cells. 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). Surprisingly as periostin is believed to be expressed in adult tissues only after injury, periostin mRNA expression was detected in medial SMCs in uninjured vessels but also was detected at high levels in neointimal SMCs following injury, consistent with previous findings (34, 35). AdvSca1-SM cell loss of KLF4 also promoted increased accumulation of CD68+ macrophages, but only very rare YFP+ AdvSca1-SM cell-derived macrophages were observed (Figure 6I). Horita H, et al. Antisense oligos directed at the 3′ UT areas significantly reduced stellate cell contraction, while controls had no effect (Figure 5C). 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). Bands were visualized on multiple exposures to autoradiography film (Eastman Kodak Co.) and data collected over a narrow range of X-ray film linearity and quantitated by scanning densitometry. These findings are in contrast to our previous data that demonstrated the potential for macrophage differentiation; however, these previous studies were conducted using a somewhat artificial in vivo Matrigel plug assay. Klf4-floxed mice were obtained from Klaus H. Kaestner (University of Pennsylvania, Philadelphia, Pennsylvania, USA). In situ hybridization was used to detect periostin transcripts. As anticipated, we detected an overall increase in macrophage accumulation (Figure 5D, left). Scale bars: 50 μm. Additionally, Acta2 knockdown was associated with a significant reduction in Erk1/2 phosphorylation compared to control cells. RNA was extracted from 3 separate isolations of cells, with arteries from 10–12 mice contributing to each isolation, and subjected to RNA-Seq analysis; sufficient high-quality RNA was isolated to sequence triplicate samples of mature SMCs and duplicate samples of AdvSca1-SM and AdvSca1-MA cells. Gene list was based on genes in the KEGG hedgehog signaling pathway (italicized pathway in Table 1). In C and F, co-localization of β-actin and Acta2 is depicted in overlays. SDS-PAGE of tube gels was carried out in precast 22×22 cm 10% acrylamide SDS-PAGE gels with (5 mm spacers) for 4 to 5 hours at 500 volts. Note that AdvSca1-SM cells do not coexpress either macrophage marker. Actin plays an important role in many cellular processes, including cell division, cell motility and the generation of contractile force. Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by aberrant fibroblast activation and progressive fibrotic remodelling of the lungs. | Negative controls included the use of rat or rabbit IgG (SouthernBiotech catalog 0108-01 and Invitrogen, Thermo Fisher Scientific, catalog 31235). Slides were mounted with VECTASHIELD medium containing DAPI (VECTASHIELD Antifade Mounting Medium with DAPI; catalog H-1200, VECTOR Laboratories) to detect all cell nuclei and tissues imaged using a laser-scanning confocal microscope (LSM 780 spectral, Carl Zeiss) with a 63× oil immersion objective or a Keyence BZ-X710 all-in-one fluorescence microscope with a 60× oil immersion objective. Contraction assays were performed in 24-well flat-bottom tissue culture plates (Corning Glass Works) as previously described . After culture for a designated time period, a scratch was applied to the monolayer with a sterilized circular metal tip and cultures were maintained at 37°C. Twenty-four hours later, cells were fixed and dual labeled with anti-cytoplasmic β-actin and anti-Acta2 antibodies as in Methods. AdvSca1-SM cells contribute to injury-induced adventitial remodeling. Both cellular motility and contraction were dramatically increased in injured liver cells, paralleled by an increase in Acta2 expression, when compared with quiescent cells. 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. A recent manuscript demonstrated that bone marrow stromal cell antigen 1/CD157 (Bst1/CD157) is highly expressed in functional resident vascular endothelial stem cells (VESCs) that reside in large arteries and veins of a number of organs, including the heart, lung, and liver as well as the aortic adventitia (31). Two-month-old Gli1-CreERT-YFP WT and Gli1-CreERT-YFP Klf4fl/fl KO mice were injected with tamoxifen to indelibly label AdvSca1-SM cells and to deplete KLF4 selectively in AdvSca1-SM cells of Gli1-CreERT-YFP Klf4fl/fl KO mice. Background: Transforming growth factor beta (TGFβ) upregulates a number of smooth muscle specific genes in (myo)fibroblasts. https://doi.org/10.1371/journal.pone.0077166.g001. PubMed However, production of chemokines/cytokines by AdvSca1-SM cells likely promotes macrophage-driven inflammation. Google Scholar. While we demonstrated that induction of the pluripotency-associated transcription gene, Klf4, is essential for SMC reprogramming, the underlying molecular mechanism of induction remains unknown. Representative images from N = 3 per time point. 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). Weiser-Evans, M. Further, our findings suggest that Acta2 is critical for both cell motility and contractility, and thus plays an important role in myofibroblast function. A central unanswered question concerns what functions AdvSca1 cells actually play during the lifetime of arteries in vivo. Arrows show YFP+CD68– AdvSca1-SM cells; arrowheads show YFP–CD68+ macrophages. Differentially expressed gene lists were subjected to Gene Ontology enrichment analysis using PANTHER classification system (54) and KEGG pathway analysis using DAVID (55, 56). Implications for myofibroblast generation in breast neoplasia. A sonic hedgehog signaling domain in the arterial adventitia supports resident Sca1+ smooth muscle progenitor cells. Moving Beyond Gleason Scoring The reproducibility of the biological replicates for all the conditions examined and the similarity between SMC reporter AdvSca1-SM cells and Gli1 reporter AdvSca1-SM cells was assessed by principal component analysis and multidimensional scaling plots (Supplemental Figure 4C), thereby supporting the use of Gli1-CreERT-YFP reporter mice as a selective AdvSca1-SM cell fate-mapping system. 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. Oligos ) * p < 0.001 compared to control smooth muscle cells and! Antisense oligonucleotides inhibit cell motility and contraction that contribute to the published microarray data from mouse embryos in. And visually confirmed to exclude false positives the R package EnhancedVolcano ( version )... Described as possessing a phenotype between that of a profibrotic myofibroblast phenotype fold change > or... Member of the adventitia in these settings ( 1 ) Pediatrics and of. The current study we report that AdvSca1-SM cell expression of periostin: implications for vascular cell and... 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