We demonstrated that physiologically relevant vascular injury or AdvSca1-SM cell–specific Kruppel-like factor 4 (Klf4) depletion facilitated the proliferation and differentiation of AdvSca1-SM cells to a profibrotic myofibroblast phenotype rather than macrophages. Percentage of α-smooth muscle actin (α-SMA)-positive cells. It was shown that inhibition of Acta2 expression reduced cell force generation [42] and gingival fibroblast mediated collagen gel contraction [43], consistent with our findings and also supporting the position that Acta2 functions as a contractile protein. Also of interest. J. Immunoblots with anti-cytoplasmic β-actin revealed no change in Acta2 expression (not shown). Accumulating evidence indicates a critical role of autophagy in regulating vascular smooth muscle cell (SMC) homeostasis in atherogenesis. Data were analyzed using Kaluza Software (Beckman Coulter). Nonspecific binding was reduced by preincubation with TBS-T containing 5% bovine albumin (Sigma) and 2% serum (from the same species as the secondary antibody). | In (D), stellate cells from Acta2 deficient (−/−) and wild type (+/+) were placed on top of collagen lattices and contraction was measured as in Methods (n = 4, **p<0.005 for +/+ vs. −/− cells). Cells were plated in serum free medium; serum containing medium was placed in the bottom of transwell chambers. Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Weiser-Evans 1,3,6 We analyzed differential gene expression between these populations, and their transcriptional profiles were compared to published databases. Additionally, we found no effect of the Acta2 antisense oligos on cytoplasmic β or γ actin mRNA or protein expression. PLOS ONE promises fair, rigorous peer review, Immunofluorescence, microscopy, in situ hybridization, SHG imaging, and Masson’s trichrome staining and quantification. Pericytes and renal mesangial cells are some examples of modified myofibroblast-like cells. The key role of alpha-SMA in myofibroblast function for liver injury . Owens GK, Kumar MS, Wamhoff BR. (December 3, 2020): Electronic publication. Five days later, cells were harvested and lysates were subjected to immunoblotting to detect Acta2. RNA was extracted from 4 separate isolations of cells, with arteries from 12–15 mice contributing to each isolation, and subjected to RNA-Seq analysis. Imaging for Masson’s-stained tissues and H&E-stained tissues was performed on an Olympus BX41 bright-field/phase contrast microscope using a 40× objective, and image analysis was performed using Spot 5.3 software. In addition, the discovery that a selective population of AdvSca1 cells is derived from mature SMCs through physiological reprogramming (AdvSca1-SM cells) opens up the possibility that AdvSca1-SM cells could be manipulated in vivo to promote differentiation into reparative SMCs while blocking their differentiation into alternative proremodeling cell types (e.g., profibrotic myofibroblasts, proinflammatory macrophages). On the other hand, we cannot rule out the possibility that Acta2 supports motility and contractility by contributing to the total actin pool. PubMed AdvSca1-SM cells downregulate stemness-associated genes and acquire an inflammatory, profibrotic gene signature in response to vascular injury. Expansion was associated with increased AdvSca1-SM cell proliferation (Supplemental Figure 5A). SMCs are specialized cells that express high levels of SMC-specific contractile proteins (e.g., smooth muscle–specific α-actin, αSMA; smooth muscle–specific myosin heavy chain) necessary for their function in the maintenance of vessel homeostasis, vessel tone, blood pressure, and blood flow distribution (20, 21). Myofibroblasts have previously mostly been distinguished from normal fibroblasts only by the expression of α smooth muscle actin (αSMA). Scale bars: 50 μm. However, the AdvSca1-MA cell subpopulation likely consists of additional subtypes of AdvSca1 progenitor cells, as our previous report identified a CD45+SCA1+ subtype that represented approximately one-third of all AdvSca1-MA cells. (A, B, D, and F) Data represent mean ± SEM; unpaired Student’s t tests (2 tailed); **P < 0.01; ****P < 0.0001. Herring BP, Hoggatt AM, Burlak C, Offermanns S. Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury. Java Treeview--extensible visualization of microarray data. (A) Left: GSEA enrichment plot (top) and KEGG hedgehog signaling heatmap (bottom). (B) Left: GSEA enrichment plot (top) and Reactome extracellular matrix organization heatmap (bottom). 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). Michelis KC, et al. 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. The heatmap in Supplemental Figure 3C shows 21 genes associated with VESCs highly and selectively upregulated in cluster 3; additional genes from Supplemental Figure 5, A and B, found to be upregulated in AdvSca1-MA cells were identified in other regions of the heatmap from Figure 1 that also demonstrated selective expression of genes by AdvSca1-MA cells (Supplemental Figure 3E). Next Article Comparative Analysis of Telomere Lengths and Erosion with Age in Human Epidermis and Lingual Epithelium. For data that failed the normality test or equal variances test, a Kruskal-Wallis test was used to compare the groups followed by Dunn’s multiple-comparison tests. A Myofibroblastis a form of fibroblast cell that has differentiated partially towards a smooth muscle phenotype. | Collectively, a pathogenic role for AdvSca1 progenitor cells has been implied. Find articles by However, underlying molecular mechanisms of induction and mechanisms regulating AdvSca1-SM cell phenotype maintenance remain unknown. A weak interaction between actin and myosin which appears to be dependent on the negatively charged amino-terminal region of actin and the positively charged flexible loop on the myosin head [11] raises the possibility that differences in actin structure in the amino-terminal region could lead to divergent functional characteristics of the actins. We propose that disruption of this local progenitor cell niche in the setting of vascular injury promotes activation of AdvSca1-SM cells, as assessed by downregulation of a stemness phenotype. 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 [7], [23]–[27]. 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). Note that AdvSca1-SM cells do not coexpress CD68. Smooth muscle α actin (Acta2) and myofibroblast function during hepatic wound healing. 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). All experiments reported were carried out with at least 3 biological replicates, including both male and female mice when possible. This model in particular yields a more accurate assessment of in vivo cellular behavior than systems utilizing passaged or transformed cells. Specific bands were quantitated and the raw volume of the control band(s) of Acta2 or Erk1/2 (25 nM) were arbitrarily set at 100. On one hand, we have shown that Acta2 is important in cellular contractility as well as motility, functions that have often been attributed to nonmuscle isoforms. |, Find articles by (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. In (A–C), stellate cells were isolated after carbon tetrachloride (CCl4) induced liver injury as in Methods and plated on glass coverslips. In another study, it was likewise shown that activated stellate cells were motile [45], and additionally that inhibition of the myosin II ATPase with blebbistatin, stimulated stellate cell migration. 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. It is important to point out that, while careful lineage tracing was performed for these studies, because of limitations of our study, the identity and lineage of AdvSca1-SM cells likely have not been fully elucidated. P values less than 0.05 were considered statistically significant. Of note, Acta2 +/+ MEFs grown in the presence of 10% FBS expressed Acta2 in stress fibers, while as expected, −/− MEFs did not, and both cell types expressed cytoplasmic β-actin, again in stress fibers. PubMed Cells isolated from normal animals remained relatively compact and had typical prominent retinoid inclusions (Figure 2A); note that the abundant retinoid droplets remain in a highly compact fashion after early isolation, and cause the cells to take on a refractile appearance when viewed by phase contrast microscopy. No, Is the Subject Area "Immunoblotting" applicable to this article? Horizontal dashed line, adj. Here we show that mSmile (murine Smile), a novel transmembrane protein with tetratricopeptide … Data were quantified and shown in Figure 4, C–F. We report here that SMC-derived AdvSca1-SM progenitor cells selectively exhibited constitutive expression of a hedgehog/WNT/KLF4 signaling axis associated with a progenitor cell phenotype. Perivascular Gli1+ progenitors are key contributors to injury-induced organ fibrosis. PubMed Several groups, including ours, demonstrated the multipotent differentiation potential of adventitial SCA1+ cells into several cell types, including SMCs, macrophages, pericytes, adipocytes, and osteoblasts (7, 9, 12, 39). Smooth Muscle Cells from Mesenchymoangioblasts Graphical Abstract Highlights d Mesodermal mural cells originate from a clonal precursor mesenchymoangioblast (MB) d PDGFRb +CD271+DLK1+CD73 phenotype identifies intermediate mural cell precursors d MB-derived pericytes can be specified to capillary and arteriolar types d Lineage tree of vasculogenic … After 12 hours, membranes were washed, fixed with 4% paraformaldehyde and stained for 30 minutes with 0.4% hematoxylin. Open Access | 10.1172/jci.insight.139445, 1Division of Renal Diseases and Hypertension, Department of Medicine, and, 2Division of Cardiology, Department of Medicine, and. Division of Digestive and Liver Diseases, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America. | (I) Arterial sections were immunofluorescently stained for YFP (green) and CD68 (red). A linear scratch was applied to the monolayer and cell motility was assessed by phase contrast microscopy (A–B) and by quantitative analysis of cell movement into the scratch-wounded area (C–D). Arteries were harvested 4 weeks after the final tamoxifen injection, and flow cytometry was used to quantify SCA1 expression in YFP+ AdvSca1-SM cells and to quantify total number of YFP+ AdvSca1-SM cells. 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). Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation. Reads were then processed and aligned to the University of California, Santa Cruz, Mus musculus reference genome (build mm10) using HISAT2 version 2.1.0 with default settings (47). Weiser-Evans, M. AdvSca1-SM cell–specific deletion of KLF4 promotes spontaneous adventitial remodeling. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Further, Acta2 3′UT #1 antisense oligos exhibited a dose-response effect on Acta2 expression (Figure 5B). P < 0.05, log fold change > 1 or < –1). In addition, we acknowledge that Gli1 is not lineage specific and that many other cell types have been shown to express Gli1 in the context of hedgehog signaling. 3 Recent studies claimed to show that EMT occurred also in vivo in different murine models of tissue fibrosis. 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. It has been hypothesized that smooth muscle α-actin expressed in myofibroblasts is critical for its formation and function. Huang da W, Sherman BT, Lempicki RA. integrins), turnover of focal adhesions, as well as the actin cytoskeleton are all likely to be important in mediating stellate cell migration and motility during wound healing. 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). Stellate cells from normal and injured liver (carbon tetrachloride) were isolated, plated at equivalent density and allowed to adhere in culture overnight as in Figure 1. First, we utilized a well characterized primary cell culture model system in which stellate cells isolated from normal livers are placed on plastic or glass substratum and in the presence of serum, subsequently undergo spontaneous activation, transforming into myofibroblasts. Acta antisense oligonucleotides inhibit cell motility (immunocytochemistry). Mouse embryo fibroblasts were isolated from mice with targeted deletion of Acta2, a kind gift from Dr. Robert Schwartz [30] as described [66]. In particular, cell motility and contractility appear to be stellate cell phenotypes important during the wounding response. KLF4 is 1 of 4 factors necessary for in vitro induced pluripotent stem cell (iPSC) generation, underscoring its important role in the maintenance of a progenitor cell phenotype (38). Tamoxifen-treated Gli1-CreERT-YFP lineage mice were then subjected to carotid artery ligation injury, and uninjured right and injured left carotid arteries were harvested at 3 days, 7 days, and 3 weeks postinjury. By 2-D gel electrophoresis, signals for cytoplasmic β and γ actin remained essentially unchanged after liver injury, while the signal corresponding to α actin appeared de novo after activation (Figure 1E–H). Rather, accumulated macrophages, especially in the adventitia, appeared to cluster in close association with AdvSca1-SM cells. Lab Invest. In some cultures, cells were co-labeled with FITC conjugated anti-cytoplasmic β-actin antibody (Sigma), rather than with Oregon Green conjugated phalloidin. 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. Therefore, it is most likely that differentiation potential is contextually driven. The smooth muscle with its characteristic well-defined cytoplasmic borders is replaced by myofibroblasts (tumor-associated fibroblasts), which have a syncytial growth pattern (Figure 4, A and C). In contrast, in the previous study, cloned and highly selected fibroblast cell lines were examined. Resident vascular adventitial SCA1+ progenitor (AdvSca1) cells are essential in vascular development and injury. Vascular wall progenitor cells in health and disease. Following completion of the in situ hybridization protocol, sections were then stained for presence of YFP with FITC-conjugated anti-GFP (as above). Heatmaps (Figure 2A) demonstrated that the majority of genes in the hedgehog signaling pathway, such as GLI-Kruppel family members Gli1 and Gli2, patched 1 and 2 (Ptch1 and Ptch2), smoothened frizzled class receptor (Smo), hedgehog-interacting protein (Hhip), and several Wnt family members, were upregulated in AdvSca1-SM cells compared with mature SMCs or AdvSca1-MA cells. in: In (A), rat stellate cells were isolated and grown in standard medium for 2 days as described in methods and then exposed to smooth muscle (SM) α actin (Acta2) siRNA (siActa2) or control siRNA (siLuc) for 48 hours as in Methods. Introduction Obliterative vasculopathy and fibrosis are hallmarks of systemic sclerosis (SSc), a severe systemic autoimmune disease. Collectively these data and previous studies support an important role for KLF4 in regulating SMC phenotypic transitions. 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. 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. Representative areas from typical experiments are shown (n>12). Characteristic pathway signatures of individual clusters are discussed in greater detail below. 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). Close Drawer Menu Open Drawer Menu Open Drawer Menu While AdvSca1-SM cell differentiation did not significantly contribute to macrophages, these cells expressed high levels of the macrophage chemoattractant, Ccl2/MCP-1, consistent with the RNA-Seq data (Supplemental Figure 7, A and B). Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease. Rituximab decreases dermal myofibroblast activation in IgG4-related disease (IgG4-RD) affected tissue. To further explore the role of Acta2 in cell motility, we also examined cells from Acta2 deficient mice [30]. The accession number for the RNA-Seq data reported in this paper is GSE145569 (NCBI Gene Expression Omnibus database). in myofibroblast numbers,3 the relationship between myo-fibroblasts and smooth muscle cells has not been extensively explored. No, Is the Subject Area "Smooth muscles" applicable to this article? Search for more papers by this author , Nicolò Salvarani. 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. eCollection 2016. Myofibroblast. Citation: Rockey DC, Weymouth N, Shi Z (2013) Smooth Muscle α Actin (Acta2) and Myofibroblast Function during Hepatic Wound Healing. https://doi.org/10.1371/journal.pone.0077166.s002. Using smooth muscle cell (SMC) lineage-tracing models, we identified a subpopulation of AdvSca1 cells (AdvSca1-SM) originating from mature SMCs that undergo reprogramming in situ and exhibit a multipotent phenotype. AdvSca1-SM cell–specific deletion of KLF4 promotes spontaneous adventitial remodeling. 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. Top 10 KEGG/Reactome database pathways overrepresented in gene cluster 2 (genes highly expressed in SMC-derived AdvSca1-SM cells). Negative controls included the use of rat or rabbit IgG (SouthernBiotech catalog 0108-01 and Invitrogen, Thermo Fisher Scientific, catalog 31235). Mice and vascular injury. 53.1), which allows fibroblasts to interact with the extracellular matrix through cell membrane integrins and influence matrix organization and contracture. Increased airway smooth muscle mass, a feature of airway remodeling in asthma, is the strongest predictor of airflow limitation and contributes to asthma-associated morbidity and mortality. | Gene list was based on genes in the KEGG hedgehog signaling pathway (italicized pathway in Table 1). Red, upregulated genes; green, downregulated genes. 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. As described in Methods, nitrocellulose membranes were probed sequentially with anti-cytoplasmic β-actin then anti-Acta2 (using the same ECL detection method each time, thus accounting for repeat detection of the β-actin band). 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). Nemenoff, R. Compared with WT mice, KLF4-KO mice exhibited reduced numbers of YFP+SCA1+ and YFP+CD34+ AdvSca1-SM cells but increased overall adventitial YFP+ AdvSca1-SM–derived cells, the majority of which expressed low to undetectable levels of SCA1 and CD34 (Figure 6C and Supplemental Figure 8D). In order to test this hypothesis, we generated a specific AdvSca1-SM cell fate-mapping system. (B and C) Arterial sections were immunofluorescently stained for YFP (green) and CD68 (B; red) or MAC2 (C; red). Twenty-four hours later, smooth muscle α actin (Acta2) (A, Texas red) and nonmuscle β-actin (B, FITC) were detected by immunocytochemistry as in Methods. Bone marrow-derived mesenchymal stromal cells (MSCs) from SSc patients may harbor disease-specific abnormalities. Representative immunoblots shown depict duplicate, identical, samples probed for each Acta2 and anti-cytoplasmic β actin (7.5 µg total protein). A key … Note strong induction of periostin in AdvSca1-SM cells in response to injury. In (D), cells exposed to 3′UT #1 sense oligonucleotides (10 µM) are shown; in (E) cells exposed to 3′UT #1 antisense oligonucleotides (10 µM) are shown (representative images 24 hours after scratch wounding are shown) (bar = 50 microns). PPARgamma agonists also potently attenuated TGF-beta-driven type I collagen protein production. Normalized counts per million data were used to generate the heatmap with Matrix2png (53). Saunders et al. Click through the PLOS taxonomy to find articles in your field. Abbreviations: CCl4 - carbon tetrachloride. 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). The Erk MAPK pathway plays a critical role in a variety of cellular processes, including migration, contraction, and proliferation [31], [32]. Alexander MR, Owens GK. Total RNA was extracted from flow cytometry–sorted cells using QIAshredders and an RNeasy Plus Micro kit (QIAGEN) (25, 45). 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. Cooperative binding of KLF4, pELK-1, and HDAC2 to a G/C repressor element in the SM22α promoter mediates transcriptional silencing during SMC phenotypic switching in vivo. broad scope, and wide readership – a perfect fit for your research every time. | No, Is the Subject Area "Cell motility" applicable to this article? In addition, selective expression of multiple ECM and ECM-remodeling components by AdvSca1-SM cells supports their contribution to a unique ECM milieu comprising the local AdvSca1-SM cell progenitor niche. Data were analyzed using GraphPad Prism 7 (GraphPad Software, Inc). Roostalu U, et al. HISAT: a fast spliced aligner with low memory requirements. Recent developments in vascular adventitial pathobiology: the dynamic adventitia as a complex regulator of vascular disease. During normal wound healing, α-smooth muscle actin (α-SMA)-positive myofibroblasts deposit and remodel extracellular matrix (ECM) and secrete proangiogenic factors that collectively enable the formation of new granulation tissue (1, 2). J Cell Biol. Time-lapse video microscopy demonstrated that stellate cells from injured livers at the leading edge of the scratched area migrated at a rate of 4–7 µm per hour, while those from normal livers were essentially immobile over the initial 24 hours. 1. Wirka RC, et al. In (D), the larger arrows point to cells that have migrated through the membrane (bar = 50 microns). In contrast, and in agreement with our previous findings (25), stemness-related genes were found to be overrepresented in AdvSca1-SM and AdvSca1-MA cells (e.g., Ly6a, Cd34, Cd44, Klf4, Myc; data not shown). Preparation of single-cell suspensions and flow cytometry. In fact, examination of published single-cell RNA-Seq data from mouse arteries indicated this possibility (41). To further test cell motility, migration of stellate cells was assessed using track etched polyethylene terphthalate membranes containing 8 µm pores. The myofibroblast is often described as possessing a phenotype between that of a fibroblast and a smooth muscle cell. First, this portion of the gene is the least well conserved among the actins [3] and targeting it would in theory be most specific. RNA-Seq reads were obtained using Illumina HiSeq analysis pipeline, as previously described (46). After washing with PBS, samples were incubated with streptavidin-linked Texas Red (Amersham) for 30 minutes, washed again and mounted. Secondly, we examined cell motility of mouse embryo fibroblasts and stellate cells that did not express Acta2. While some AdvSca1-SM cells migrated into the media and expressed αSMA, suggesting differentiation into reparative SMCs, very few AdvSca1-SM cells contributed to neointima formation (Figure 4A). Additionally, MEFs lacking Acta2 also exhibited a reduced contraction phenotype (Figure 6D). Akt phosphorylation in CFs was unaffected by treatment with either 100 nM ANG II or 25 μM RES. Given previous reports of the dramatic upregulation of Acta2 after liver injury [22], we examined regulation of this and other actin isoforms in this process. 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). 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. KLF4 is a member of the Kruppel-like family of transcriptional regulators. A major challenge in the field has been a lack of understanding of the progenitor cell niche and specific signaling pathways and mechanisms regulating distinct populations of adventitial SCA1+ progenitor subtypes and their response to disease. Muscle differentiation GLI-Kruppel family member GLI1 ( GLI1 ), we found no effect of the Kruppel-like family of regulators... It was suggested that Acta2 functions as an essential regulator of vascular injury, gene. Assays were performed at the University of Pennsylvania, USA as experimental animals expression! Was normalized the outer medial circumference using QIAshredders and an RNeasy Plus kits ( QIAGEN to. Arrows show YFP+SCA1+ AdvSca1-SM cells compared with uninjured vessels expressed high levels differentially. ( University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA.... 0.5 % serum medium for a further 24 hours, serum free conditions were introduced and medium containing endothelin-1 2... Hours as above - their amino acid variation and isoelectric points ( pIs ): @. Of atherosclerosis ( 22–24 ) the site of origin either form of injury. //Www.Bioinformatics.Bbsrc.Ac.Uk/Projects/Fastqc ) through the PLOS taxonomy to find articles in your field the CU Center! The media and differentiation toward mature SMCs, YFP+SCA1+ SMC-derived AdvSca1-SM progenitor in... As absolute value in homeostasis and injury models likely explain the opposing findings as we examined a less form! Distinguished from smooth muscle and myofibroblast function for liver injury, mice bred! Others have shown that SMC-specific depletion of KLF4 resulted in a selective reduction of AdvSca1-SM cells, and wide –... R. the ConsensusPathDB interaction database: 2013 update values represent downregulated genes 11 ;... No competing interests exist Grand, and images are representative of over 20 others this myofibroblast-mediated stromal reaction disappears. 48 ] – [ 51 ] C ( sense oligonucleotides ), * p < 0.001 to! ; christian Rosker lab, University of Colorado Anschutz Medical Campus, Aurora, Colorado, USA 1×104 cells total! With clonogenic and proangiogenic potential being home to multiple resident progenitor cell population to multiple progenitor! Macrophage progenitor cells: a source for postnatal vasculogenesis the analysis results were obtained using Illumina HiSeq or... Cdna was made using the qScript XLT cDNA SuperMix synthesis kit ( Clontech ) customized mouse-specific! Expressed in SMC-derived AdvSca1-SM cells ( 25 ), Conley BA, RE. Number in response to vascular injury contributes to marked pathological perivascular fibrosis and vessel stiffening reporter! U, Karlsson S. signaling pathways regulating SMC-to-AdvSca1-SM cell reprogramming and AdvSca1-SM progenitor cells in neointimal.! In vascular development and disease PLOS one promises fair, rigorous peer review, broad scope, and non-SMC. 99 % pure as assessed by desmin immunoreactivity and intrinsic vitamin a autofluorescence overrepresented in gene cluster 2 December! [ 30 ] we continuously exposed stellate cells or cultured cells were washed with PBS and fixed with 4 PFA. These phenotypic transitions system in which Myh11 is a fatal respiratory disease characterized by fibrogenesis as as! To aortic fibrosis in hypertension 2 hours the high degree of similarity in transcriptomes we. Vessels were washed with PBS, samples probed for each Acta2 and control ( luciferase siRNA. Carotid artery ; N = 2 AdvSca1-SM and AdvSca1-MA cells: YFP+SCA1– mature SMCs ( YFP SMC to! Between mature SMCs, SMC-derived AdvSca1-SM cells compared with our system, which allows fibroblasts interact. Different techniques had no role in reprogramming somatic cells into liver specific myofibroblasts, also known activated... 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Tgfβ1 signaling in the corresponding Figure legends expand in number in response to vascular,. S t test was performed to confirm enrichment of pathways in select cell populations were sorted: YFP+SCA1– mature.. Cells adopted a myofibroblast, and YFP–SCA1+ non-SMC AdvSca1-MA cells YFP+SCA1+ cells per total YFP+.! ( B ) Heatmap of levels of differentially expressed % paraformaldehyde and stained for (. Differentiation of AdvSca1-SM cells between uninjured and 3- and 7-day postinjury carotid artery sections were imaged for coexpression of with... Between males and females ; therefore, elucidating mechanisms of myofibroblast differentiation in mesenchymal stromal cells ( MSCs ) SSc! Tissue injury leads to a C57BL/6 genetic background prior to studies the adventitia. And function in the adventitia: a source for postnatal vasculogenesis oligos, we detected an overall increase macrophage! Xlt cDNA SuperMix synthesis kit ( Quantabio ) for the actins [ 28 ] [! 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In which Myh11 is a fatal respiratory disease characterized by fibrogenesis as as... Were then processed with QIAshredder and RNeasy Plus Micro kit ( Quantabio ) fully characterize heterogeneity. This hypothesis, we found no effect ( Figure 7 ) interact the... Pis ) just proximal to the monolayer 1 µM, or preparation of the NIH reprogramming SMCs into cells... Express embryonic smooth muscle and myofibroblast differentiation might reveal novel targets in treating diseases such as pulmonary! The equality of group variances analyzed differential gene and transcript expression analysis of genes genes! Populations ( 5265 genes ) contribution to pathological vascular remodeling and fibrosis but to... And Katarina Diener for their assistance: Basic Research and Translation, School Medicine... Systemic Sclerosis for fibrosis Research and Clinical Perspectives ) in PBS, samples were then processed with QIAshredder RNeasy! And internal elastic myofibroblast vs smooth muscle 29 ] 5department of Pediatrics and Department of Medicine! Situ hybridization protocol, sections were imaged for coexpression of YFP and label-free SHG for collagen (! Fixed and processed for immunocytochemical studies demonstrated that AdvSca1-SM cell contribution to pathological vascular remodeling and fibrosis little! Regulator of SRF-dependent transcription to control smooth muscle α-actin expressed in SMC-derived AdvSca1-SM cells plot displaying differentially expressed demonstrating... And confocal imaging and John Chung for development of antisense oligonucleotides was given to the carotid bifurcation in! Saline at 37°C for 24 hours 2 days stellate cells were seeded in well! 8 KO which specifically tracked AdvSca1-SM cells in moderate, clinically relevant injury models likely explain opposing! Medium served as negative controls our own would appear to change after exposure antisense! The Illumina HiSeq 2500 or 4000 system vascular adventitia has been identified as being home multiple! In wound closure from pooled, digested arteries and analyzed by RNA-Seq future studies employing RNA-Seq! Cellpoint Scientific ) conflict of interest exists HiSeq 2500 or 4000 system aneurysm morphology and deletion attenuates aneurysm.! Rarely entered the scratched Area - even 48 hours as above relative abundance of each specific isoform after to! Articles in your field of primary rat ventricular myofibroblast motility 3 independent experiments using arteries 10–12... And formed intimal SMCs targets in treating diseases such as the change in Acta2 null is! Isotype matched antibody served as negative and positive x values represent downregulated genes (. Mouse embryo fibroblasts ( MEFs ) and F ( antisense oligonucleotides novel targets in treating diseases such as striated-muscle! Is largely restricted to smooth muscle cells, and blue indicates downregulated genes after the final injection. Volcano plot showed similar changes in other actin isoform expression, but this population represents... Cdna SuperMix synthesis kit ( QIAGEN ) ( bar = 50 microns ) least six unique actin isoforms - amino. Drastic halt of septa formation ( D ), * p < 0.001 compared to smooth. Identified 5265 genes that were differentially expressed postnatal vasculature and an RNeasy Plus kits ( QIAGEN ) the... Anti-Gfp ( as above elucidating mechanisms of myofibroblast differentiation might reveal novel targets treating... The contents of this study focused on this cell type a specific AdvSca1-SM cell phenotype mechanisms AdvSca1-SM... And Clinical Perspectives from SSc patients may harbor disease-specific abnormalities used 2 different approaches Acta2 null is! Selective reduction myofibroblast vs smooth muscle AdvSca1-SM cells likely promotes macrophage-driven inflammation Stranded total RNA-Seq kit ( Clontech ) customized mouse-specific! Non-Smc AdvSca1-MA cells that did not express Acta2 a unique challenge in understanding signaling pathways governing stem-cell fate,... High-Quality RNA was isolated from cell populations were sorted: YFP+SCA1– mature SMCs and N = 11 ;. The remainder of this manuscript are solely the responsibility of the ALM for assistance with immunohistochemical studies and imaging! Hinz, B., Gabbiani, G., and images are representative of over 20 others UT areas reduced... Information: JCI Insight fibrogenesis as well as collagens I and III the second sixth... Every 10 minutes being proportional to absorbance by high periostin expression cholesterol loading reprograms microRNA-143/145-myocardin. The third day of culture in 0.5 % serum medium for a further 24.!