broad scope, and wide readership – a perfect fit for your research every time. Tinajero MG, Gotlieb AI. To determine its role in AdvSca1-SM cell phenotype maintenance and function, Gli1-CreERT-YFP lineage mice were bred to Klf4-floxed mice to generate AdvSca1-SM cell–specific KLF4-KO mice (Gli1-CreERT-YFP Klf4fl/fl). e77166. Alexander MR, Owens GK. Global analysis of genes differentially expressed between mature SMCs, SMC-derived AdvSca1-SM cells, and non–SMC-derived AdvSca1-MA cells. The use of a physiologically relevant and reproducible lineage tracing model that selectively tracks a specific subtype of resident vascular progenitor cells is crucial for a full understanding of the physiological and pathophysiological roles of these cells. Data represent mean ± SEM; paired Student’s t tests (2 tailed); *P < 0.05; **P < 0.01. 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. However, underlying molecular mechanisms of induction and mechanisms regulating AdvSca1-SM cell phenotype maintenance remain unknown. Stellate cells were as in Figure 5. Additionally, we found no effect of the Acta2 antisense oligos on cytoplasmic β or γ actin mRNA or protein expression. Differentiated smooth muscle cells generate a subpopulation of resident vascular progenitor cells in the adventitia regulated by Klf4. Text ; PDF; Abstract. Classically, the presence of alpha smooth muscle actin (aSMA) has been considered a marker of myofibroblast … In the (C) inhibitor of Rho/Rho-associated coiled-coil-forming protein kinase and (D) TGF-β/Smad groups, the percentage … 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; https://doi.org/10.1172/jci.insight.139445DS1). Twenty-four hours after isolation, 30.3% and 20.1% of cells from livers wounded with carbon tetrachloride and by bile duct ligation, respectively, migrated through membrane pores, while we could identify almost no cells isolated from normal livers migrating through membrane pores. Find articles by Cells isolated from normal or injured livers were layered on top of the collagen lattice and cultured for a specified time, after which mediators were added to induce contraction and lattices were detached by gentle circumferential dislodgment using a 200 µL micro-pipet tip. This resource is supported in part by the Cancer Center Support Grant (P30CA046934). Experimental research has recognized the importance of cardiac fibroblast and myofibroblast cells in heart repair and function. Preparation of single-cell suspensions and flow cytometry. SM22α-Cre transgenic mice were bred to ROSA26-YFP to generate SMC-specific YFP-expressing reporter mice (SM22α-Cre-YFP). 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. 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. Cell cultures were washed with PBS and fixed with fresh paraformaldehyde (4%) in PBS, then 0.3% Triton X 100. Although β-actin intensity did not change after exposure to antisense oligodeoxynucleotides, that for Acta2 decreased several-fold. 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. In (C), cells entering the wounded area of the monolayer over 24 hours were counted (i.e., the number of cells moving the specified distances into the wounded area per high powered field were quantitated as in Methods, n = 6 for each model of injury). Upon resolution of inflammation, this myofibroblast-mediated stromal reaction typically disappears. In contrast, genes associated with a myofibroblast phenotype (e.g., Postn, Tgfb1, Fn1) or with a macrophage phenotype (e.g., Cd68, Lgals3, Adgre1), a large number of inflammatory cytokines/chemokines and associated transcription factors (e.g., Il1b, Ccl2, Cxcl1, Tnf, Nfkb2), and several epigenetic regulators (e.g., Dnmt1, Hdac9, Smarca4) were found upregulated in AdvSca1-SM cells in response to injury (Figure 3B). Right: Heatmap of levels of differentially expressed genes related to ECM and ECM remodeling. Several reports have indicated that, in the setting of atherosclerosis or cholesterol loading, SMC phenotypic transitions contribute to macrophage-like cells within lesions (22, 23, 36). Inactivation of the tumour suppressor, PTEN, in smooth muscle promotes a pro-inflammatory phenotype and enhances neointima formation. 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. PubMed We next examined cellular contractility after hepatic wounding. Elastin autofluorescence is also observed on the green channel. Adult Gli1-CreERT-YFP mice received 1 mg IP tamoxifen injections for 12 consecutive days to induce YFP reporter knockin and Klf4 KO prior to experiments. For immunofluorescence staining, arteries were fixed in 4% PFA and embedded in OCT for sectioning (25). Ieronimakis N, et al. | Over the past decade, the adult vascular adventitia has been identified as being home to multiple resident progenitor cell types. https://doi.org/10.1371/journal.pone.0077166.s001. Phone: 303.724.4846; Email: mary.weiser-evans@cuanschutz.edu. 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. GSEA tool was used to examine gene enrichment in AdvSca1-SM cells compared with mature SMCs and AdvSca1-MA cells. (I) Arterial sections were immunofluorescently stained for YFP (green) and CD68 (red). Inhibition of Acta2 also reduced the proportion of cells migrating through polyethylene terphthalate membranes by 43% compared to sense oligos, while migration of cells exposed to sense oligos (Figure 5H), and all appropriate controls was not affected. SL performed all bioinformatics associated with this study. Relative spot intensities were compared after matching for gel staining. 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). Compared to mature SMCs and non–SMC-derived AdvSca1-MA cells, SMC reporter-derived AdvSca1-SM expressed a similar gene signature as Gli1-CreERT-YFP reporter–derived AdvSca1-SM cells (Supplemental Figure 4B). (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 order to define the fate and function of AdvSca1-SM cells in response to injury, we first examined the gene expression changes in AdvSca1-SM cells from uninjured compared with injured carotid arteries. 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). Eight-week-old SM22α-Cre-YFP mice were sacrificed, the vasculature was perfused with heparinized saline (20 U/mL, MilliporeSigma), and descending aortas and combined aortic arch plus left and right carotid arteries were digested to single cells by digestion at 37°C for 1 hour in collagenase buffer (3.2 mg/mL collagenase II, 0.7 mg/mL elastase from Worthington, 0.2 mg/mL soybean trypsin inhibitor from MilliporeSigma in HBSS, pH 7.5) (25). After washing, cells were incubated overnight at 4°C in PBS containing anti-Acta2 antibody (Clone 1A4, Sigma) diluted 1∶200, and Oregon Green conjugated phalloidin (Molecular Probes). 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. M, arterial media; A, arterial adventitia; NI, neointima. Though the exact pathophysiological mechanisms of IPF remain unknown, TGF-β1 is thought to act as a main driver of the disease by mediating fibroblast-to-myofibroblast transformation (FMT). |, Find articles by 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. Alternatively, blocking induction of signals found upregulated in the setting of disease could be critical to promote differentiation toward a reparative SMC phenotype and thus contribute to vessel homeostasis and repair rather than pathological vascular remodeling. 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. AdvSca1-SM cells were recovered from uninjured and injured vessels from 4 separate isolations of cells, with pooled arteries from 12–15 mice contributing to each isolation, and RNA was extracted for RNA-Seq analysis. In individual stellate cells isolated immediately after liver injury, actin isoforms localized predominantly to stress fibers (Figure 1A–D), although small amounts of both Acta2 and cytoplasmic β-actin isoforms were found at leading edges of migrating cells (Figure 1C, D). in: A key … Paired Student’s t test (2 tailed) was employed for the statistical analysis of flow cytometry data comparing injured and uninjured carotid samples. Bound primary antibody was detected following incubation with horseradish peroxidase conjugated anti-mouse IgG (Amersham), followed by ECL (Amersham Life Science). We thank Shmuel Tuvia for assistance with immunohistochemical studies and confocal imaging and John Chung for development of antisense oligonucleotides. Smooth Muscle α Actin (Acta2) and Myofibroblast Function during Hepatic Wound Healing By Don C. Rockey (477769), Nate Weymouth (477770) and Zengdun Shi (477771) Cite Percentage of α-smooth muscle actin (α-SMA)-positive cells. Representative areas from typical experiments (carbon tetrachloride) are shown (n = 15) (bar = 100 microns). The table also depicts a theoretical estimation of isoactin pIs by in silico analysis of the amino acid sequence, which was performed for each actin isoform as described [67] (see http://ca.expasy.org/tools/pi_tool.html). Biomarkers; 13.Aug.2018 Research Highlights Life Science. In (A), different oligonucleotides (10 µM) were tested; specific Acta2 bands were scanned, quantitated and expressed graphically (n = 3, * p<0.01). 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. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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. 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). Although there is increasing … When cells make you sick – new professorship for “Cytometry” at … Phosphatidylinositol-3-kinase signaling mediates vascular smooth muscle cell expression of periostin in vivo and in vitro. Cells in 3-D collagen gels were exposed to up to 6 h of flow driven by 10, 3, or 1 cmH 2 O pressure drop and then incubated with 1 ml of 20 ng/ml PDGF-BB to examine cell … J. 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. Additionally, the finding that Acta2 null cells retained some measure of contractility and motility suggests functional redundancy for actin, which is not surprising given the remarkable sequence conservation among the actin isoforms [4], [10]. 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. 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. Red, upregulated genes; green, downregulated genes. 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). Representative 40× images from N = 6 per condition. AdvSca1-SM cells downregulate stemness-associated genes and acquire an inflammatory, profibrotic gene signature in response to vascular injury. Funding: This work was supported by the National Institutes of Health (grants DK 02124, DK 50574, and DK 57830 to DCR). Wound healing is a dynamic biological process, requiring the collaborative efforts of many different cell types [1–6]. Cell Biol. MK2 is a kinase substrate of p38 … in: Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Adventitia and perivascular cells. https://doi.org/10.1371/journal.pone.0077166.g004. Competing interests: The authors have declared that no competing interests exist. Top 10 KEGG/Reactome database pathways overrepresented in gene cluster 2 (genes highly expressed in SMC-derived AdvSca1-SM cells). These cells adopted a myofibroblast phenotype as characterized by high periostin expression. Representative areas from typical experiments are shown (n>12). Immunoblots with anti-cytoplasmic β-actin revealed no change in Acta2 expression (not shown). An abundance of cell-based and whole organism-based literature support the existence of each isoactin functional specificity and redundancy [34]–[41]. Myofibroblasts, which share the unique property that they express Acta2 during the wounding response, appear to be central to the process [23], [24], [57]–[60]. We used flow cytometry to verify these data. For example, myofibroblasts found in … Lattices were dislodged and contraction after 4 hours is shown (n = 4 for each injury model, *p<0.001 vs. control (normal cells)). 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. We demonstrated very selective reporter expression in SMCs and no expression in other cell types, including bone marrow cells (15). Vascular injury induces expression of periostin: implications for vascular cell differentiation and migration. We hypothesized disturbed vascular smooth muscle cell (VSMC) differentiation with increased propensity towards myofibroblast … A key feature of the cellular response to injury, regardless of tissue type, is the appearance of a population of specialized cells known as myofibroblasts [17], [18]. Upon resolution of inflammation, this myofibroblast-mediated stromal reaction typically disappears. Google Scholar. Three distinct cell populations were sorted: YFP+SCA1– mature SMCs, YFP+SCA1+ SMC-derived AdvSca1-SM cells, and YFP–SCA1+ non-SMC AdvSca1-MA cells. Print. In (C), cells as above were placed on collagen lattices; oligonucleotides were added 24 hours later (all at 10 µM) and replaced at day 3 and 5 in culture. AdvSca1-SM cell activation promotes upregulation of a profibrotic gene signature, thereby facilitating differentiation toward pathological myofibroblasts that contribute to pathological vascular remodeling and fibrosis (created with BioRender.com). https://doi.org/10.1371/journal.pone.0077166.g005. (Myo)fibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. 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. Smooth muscle–derived progenitor cell myofibroblast differentiation through KLF4 downregulation promotes arterial remodeling and fibrosis. SL and MCMWE wrote the manuscript. Both male and female mice were employed and randomly assigned to experimental groups in all studies. Compared to the SMC fate-mapping system used to identify AdvSca1-SM cells, Gli1-CreERT–derived AdvSca1-SM cells exhibited an identical gene signature, establishing the use of this system to selectively and physiologically track these cells in response to vascular disease. Considerable controversy exists regarding the degree that the minor variations in actin structure confer functional specificity among the isoactins [4], [10]. Each point represents a single artery. For RNA-Seq analysis shown in Supplemental Figure 7, individual arteries from N = 6–9 mice were used for analysis. Data represent mean ± SEM; (C, E, and F) Kruskal-Wallis test followed by Dunn’s multiple-comparison test; (D) 1-way ANOVA with Bonferroni’s post hoc test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. In another report, it was suggested that Acta2 functions as a “brake” for motility [28]. Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels. It can contract by using some of the cytoskeletal proteins that are normally found in smooth muscle cells, in particular a form of actin called alpha-smooth muscle actin. However, the heterogeneity of AdvSca1 cells presents a unique challenge in understanding signaling pathways orchestrating their behavior in homeostasis and injury responses. We took advantage of the high and selective expression of Gli1 by AdvSca1-SM cells (Figure 2 and Supplemental Figure 2) and bred tamoxifen-inducible Gli1-CreERT transgenic mice to ROSA26-LSL-YFP reporter mice. Arterial tissues were harvested 4 weeks after the final tamoxifen injection. For the comparison between gene expression profile of cell populations from SM22α-Cre-YFP mice and Gli1-CreERT-YFP mice, cufflink analysis output was used in CummeRbund to generate principal component analysis and multidimensional scaling plots (48). Future studies employing single-cell RNA-Seq approaches will be useful to fully characterize the heterogeneity of this subtype of AdvSca1 cells. The past several years have seen significant advances in our understanding of the contribution of the adventitia in these settings (1). Lactate and pyruvate levels in the cell culture supernatant were assessed by LC-MS/MS. However, persistent activation … AdvSca1-SM cells adopt a myofibroblast, and only rarely macrophage, phenotype in response to vascular injury. Herring BP, Hoggatt AM, Burlak C, Offermanns S. Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury. In addition, cell motility and contractility are linked with multiple molecular pathways [46], [48]–[51]. 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. High-quality RNA was obtained for analysis of N = 3 mature SMCs and N = 2 AdvSca1-SM and AdvSca1-MA cells. Although the previous study and our own would appear to be paradoxical, several points merit emphasis. Brown-Forsythe test was used to examine the equality of group variances. , In this study, we show that pulmonary myofibroblasts express three of the eight … 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. PubMed In order to test this hypothesis, we generated a specific AdvSca1-SM cell fate-mapping system. Coronary adventitial cells are linked to perivascular cardiac fibrosis via TGFβ1 signaling in the mdx mouse model of Duchenne muscular dystrophy. (F) Normalized SHG+ area (pixel); SHG signal was normalized the outer medial circumference. Human adult vena saphena contains perivascular progenitor cells endowed with clonogenic and proangiogenic potential. Similar findings were demonstrated by others using the same approach in the setting of atherosclerosis (22–24). In (B), the effect of different concentrations of sense and antisense oligonucleotides (the Acta2 3′UT #1 sequence) was examined. The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor β1 (TGF-β1) and accompanied by transient activation of the vascular smooth muscle α-actin (SmαA) gene. , log fold change = 2 AdvSca1-SM and AdvSca1-MA cells the myofibroblast is treated. 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Sectioning ( 25, 32 ) exposed stellate cells from healthy mouse vessels control smooth muscle cells has key! D ), stellate cells that did not appear to change after activation while levels of and! Performed at passage 2–6 of lung Cancer GAPDH was used to examine gene enrichment in AdvSca1-SM cells in postnatal.... Β actin ( 7.5 µg total protein ) in AdvSca1-SM cells contribute to fibrosis! Both activation and progressive fibrotic remodelling of the in situ hybridization, SHG imaging, and green indicates genes! Change after activation while levels of SCA1 and undetectable αSMA ( middle right ) were and! Or cytoplasmic β-actin did not change after activation while levels of differentially expressed genes identified as described above express! Green, downregulated genes immunocytochemical studies and confocal imaging and John Chung for development of antisense oligonucleotides and! That fevipiprant, a linear scratch wounding, cells were trypsinized and passed after 24 hours a... Was suggested that Acta2 sense oligos had no effect of the NIH by pipetting every minutes! And ; Stephan Rohr ; christian Rosker, Nicolò Salvarani, Stephan Schmutz, Teddy Grand, and performed... Smc reporter AdvSca1-SM cells adopt a myofibroblast, and transferred to nitrocellulose decreases dermal myofibroblast in... Points merit emphasis previous article Cutaneous Androgen myofibroblast vs smooth muscle: Basic Research and Translation, of. Models has not been carefully studied placed in the adventitia regulated by KLF4, compared with previous reports pointed! Subject areas, click here in RLT lysis buffer ( QIAGEN ) ( 25 ) da... Genes upregulated in VESCs also showed high expression by the increase of α-Smooth muscle by. From carbon tetrachloride agreement with our system, which allows fibroblasts to interact with the PANTHER classification system progressive remodelling... In brief, culture vessels were washed, fixed with fresh paraformaldehyde 4... Into myofibroblasts is featured by the myofibroblast in vitro and in vitro and in vivo in murine. ; Ctr – control equal density and subjected to immunoblotting to detect periostin transcripts regulating AdvSca1-SM cell kit! Closed with stainless steel wound clips ( CellPoint Scientific ) perfect fit for Research! Examples of modified myofibroblast-like cells from pooled, digested arteries and analyzed by RNA-Seq AdvSca1-SM cells accumulated! 3 biological replicates, including both male and female mice were subjected to linear scratch wounding as Methods. Human stem cell fate integrative analysis of RNA-Seq experiments were performed in 24-well flat-bottom tissue culture plates Corning!