Tumor Biomechanics Alters Metastatic Dissemination of Triple Negative Breast Cancer via Rewiring Fatty Acid Metabolism.

In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.

Development of a robust induced pluripotent stem cell atrial cardiomyocyte differentiation protocol to model atrial arrhythmia.

BACKGROUND: Atrial fibrillation is the most common arrhythmia syndrome and causes significant morbidity and mortality. Current therapeutics, however, have limited efficacy. Notably, many therapeutics shown to be efficacious in animal models have not proved effective in humans. Thus, there is a need for a drug screening platform based on human tissue. The aim of this study was to develop a robust protocol for generating atrial cardiomyocytes from human-induced pluripotent stem cells. METHODS: A novel protocol for atrial differentiation, with optimized timing of retinoic acid during mesoderm formation, was compared to two previously published methods. Each differentiation method was assessed for successful formation of a contractile syncytium, electrical properties assayed by optical action potential recordings and multi-electrode array electrophysiology, and response to the G-protein-gated potassium channel activator, carbamylcholine. Atrial myocyte monolayers, derived using the new differentiation protocol, were further assessed for cardiomyocyte purity, gene expression, and the ability to form arrhythmic rotors in response to burst pacing. RESULTS: Application of retinoic acid at day 1 of mesoderm formation resulted in a robust differentiation of atrial myocytes with contractile syncytium forming in 16/18 differentiations across two cell lines. Atrial-like myocytes produced have shortened action potentials and field potentials, when compared to standard application of retinoic acid at the cardiac mesoderm stage. Day 1 retinoic acid produced atrial cardiomyocytes are also carbamylcholine sensitive, indicative of active Ikach currents, which was distinct from ventricular myocytes and standard retinoic addition in matched differentiations. A current protocol utilizing reduced Activin A and BMP4 can produce atrial cardiomyocytes with equivalent functionality but with reduced robustness of differentiation; only 8/17 differentiations produced a contractile syncytium. The day 1 retinoic acid protocol was successfully applied to 6 iPSC lines (3 male and 3 female) without additional optimization or modification. Atrial myocytes produced could also generate syncytia with rapid conduction velocities, > 40 cm s-1, and form rotor style arrhythmia in response to burst pacing. CONCLUSIONS: This method combines an enhanced atrial-like phenotype with robustness of differentiation, which will facilitate further research in human atrial arrhythmia and myopathies, while being economically viable for larger anti-arrhythmic drug screens.

Single-cell transcriptome dynamics of the autotaxin-lysophosphatidic acid axis during muscle regeneration reveal proliferative effects in mesenchymal fibro-adipogenic progenitors.

Lysophosphatidic acid is a growth factor-like bioactive phospholipid recognising LPA receptors and mediating signalling pathways that regulate embryonic development, wound healing, carcinogenesis, and fibrosis, via effects on cell migration, proliferation and differentiation. Extracellular LPA is generated from lysophospholipids by the secreted hydrolase-ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2; also, AUTOTAXIN/ATX) and metabolised by different membrane-bound phospholipid phosphatases (PLPPs). Here, we use public bulk and single-cell RNA sequencing datasets to explore the expression of Lpar 1-6, Enpp2, and Plpp genes under skeletal muscle homeostasis and regeneration conditions. We show that the skeletal muscle system dynamically expresses the Enpp2-Lpar-Plpp gene axis, with Lpar1 being the highest expressed member among LPARs. Lpar1 was expressed by mesenchymal fibro-adipogenic progenitors and tenocytes, whereas FAPs mainly expressed Enpp2. Clustering of FAPs identified populations representing distinct cell states with robust Lpar1 and Enpp2 transcriptome signatures in homeostatic cells expressing higher levels of markers Dpp4 and Hsd11b1. However, tissue injury induced transient repression of Lpar genes and Enpp2. The role of LPA in modulating the fate and differentiation of tissue-resident FAPs has not yet been explored. Ex vivo, LPAR1/3 and ENPP2 inhibition significantly decreased the cell-cycle activity of FAPs and impaired fibro-adipogenic differentiation, implicating LPA signalling in the modulation of the proliferative and differentiative fate of FAPs. Together, our results demonstrate the importance of the ENPP2-LPAR-PLPP axis in different muscle cell types and FAP lineage populations in homeostasis and injury, paving the way for further research on the role of this signalling pathway in skeletal muscle homeostasis and regeneration, and that of other organs and tissues, in vivo.

Hif-1a suppresses ROS-induced proliferation of cardiac fibroblasts following myocardial infarction.

We report that cardiac fibroblasts (CFs) and mesenchymal progenitors are more hypoxic than other cardiac interstitial populations, express more hypoxia-inducible factor 1α (HIF-1α), and exhibit increased glycolytic metabolism. CF-specific deletion of Hif-1a resulted in decreased HIF-1 target gene expression and increased mesenchymal progenitors in uninjured hearts and increased CF activation without proliferation following sham injury, as demonstrated using single-cell RNA sequencing (scRNA-seq). After myocardial infarction (MI), however, there was ∼50% increased CF proliferation and excessive scarring and contractile dysfunction, a scenario replicated in 3D engineered cardiac microtissues. CF proliferation was associated with higher reactive oxygen species (ROS) as occurred also in wild-type mice treated with the mitochondrial ROS generator MitoParaquat (MitoPQ). The mitochondrial-targeted antioxidant MitoTEMPO rescued Hif-1a mutant phenotypes. Thus, HIF-1α in CFs provides a critical braking mechanism against excessive post-ischemic CF activation and proliferation through regulation of mitochondrial ROS. CFs are potential cellular targets for designer antioxidant therapies in cardiovascular disease.

Origins, potency, and heterogeneity of skeletal muscle fibro-adipogenic progenitors-time for new definitions.

Striated muscle is a highly plastic and regenerative organ that regulates body movement, temperature, and metabolism-all the functions needed for an individual's health and well-being. The muscle connective tissue's main components are the extracellular matrix and its resident stromal cells, which continuously reshape it in embryonic development, homeostasis, and regeneration. Fibro-adipogenic progenitors are enigmatic and transformative muscle-resident interstitial cells with mesenchymal stem/stromal cell properties. They act as cellular sentinels and physiological hubs for adult muscle homeostasis and regeneration by shaping the microenvironment by secreting a complex cocktail of extracellular matrix components, diffusible cytokines, ligands, and immune-modulatory factors. Fibro-adipogenic progenitors are the lineage precursors of specialized cells, including activated fibroblasts, adipocytes, and osteogenic cells after injury. Here, we discuss current research gaps, potential druggable developments, and outstanding questions about fibro-adipogenic progenitor origins, potency, and heterogeneity. Finally, we took advantage of recent advances in single-cell technologies combined with lineage tracing to unify the diversity of stromal fibro-adipogenic progenitors. Thus, this compelling review provides new cellular and molecular insights in comprehending the origins, definitions, markers, fate, and plasticity of murine and human fibro-adipogenic progenitors in muscle development, homeostasis, regeneration, and repair.

PDGF-PDGFR network differentially regulates the fate, migration, proliferation, and cell cycle progression of myogenic cells.

Platelet-derived growth factors (PDGFs) regulate embryonic development, tissue regeneration, and wound healing through their binding to PDGF receptors, PDGFRα and PDGFRß. However, the role of PDGF signaling in regulating muscle development and regeneration remains elusive, and the cellular and molecular responses of myogenic cells are understudied. Here, we explore the PDGF-PDGFR gene expression changes and their involvement in skeletal muscle myogenesis and myogenic fate. By surveying bulk RNA sequencing and single-cell profiling data of skeletal muscle stem cells, we show that myogenic progenitors and muscle stem cells differentially express PDGF ligands and PDGF receptors during myogenesis. Quiescent adult muscle stem cells and myoblasts preferentially express PDGFRß over PDGFRα. Remarkably, cell culture- and injury-induced muscle stem cell activation altered PDGF family gene expression. In myoblasts, PDGF-AB and PDGF-BB treatments activate two pro-chemotactic and pro-mitogenic downstream transducers, RAS-ERK1/2 and PI3K-AKT. PDGFRs inhibitor AG1296 inhibited ERK1/2 and AKT activation, myoblast migration, proliferation, and cell cycle progression induced by PDGF-AB and PDGF-BB. We also found that AG1296 causes myoblast G0/G1 cell cycle arrest. Remarkably, PDGF-AA did not promote a noticeable ERK1/2 or AKT activation, myoblast migration, or expansion. Also, myogenic differentiation reduced the expression of both PDGFRα and PDGFRß, whereas forced PDGFRα expression impaired myogenesis. Thus, our data highlight PDGF signaling pathway to stimulate satellite cell proliferation aiming to enhance skeletal muscle regeneration and provide a deeper understanding of the role of PDGF signaling in non-fibroblastic cells.

Evolving Roles of Muscle-Resident Fibro-Adipogenic Progenitors in Health, Regeneration, Neuromuscular Disorders, and Aging.

Normal skeletal muscle functions are affected following trauma, chronic diseases, inherited neuromuscular disorders, aging, and cachexia, hampering the daily activities and quality of life of the affected patients. The maladaptive accumulation of fibrous intramuscular connective tissue and fat are hallmarks of multiple pathologies where chronic damage and inflammation are not resolved, leading to progressive muscle replacement and tissue degeneration. Muscle-resident fibro-adipogenic progenitors are adaptable stromal cells with multilineage potential. They are required for muscle homeostasis, neuromuscular integrity, and tissue regeneration. Fibro-adipogenic progenitors actively regulate and shape the extracellular matrix and exert immunomodulatory functions via cross-talk with multiple other residents and non-resident muscle cells. Remarkably, cumulative evidence shows that a significant proportion of activated fibroblasts, adipocytes, and bone-cartilage cells, found after muscle trauma and disease, descend from these enigmatic interstitial progenitors. Despite the profound impact of muscle disease on human health, the fibrous, fatty, and ectopic bone tissues' origins are poorly understood. Here, we review the current knowledge of fibro-adipogenic progenitor function on muscle homeostatic integrity, regeneration, repair, and aging. We also discuss how scar-forming pathologies and disorders lead to dysregulations in their behavior and plasticity and how these stromal cells can control the onset and severity of muscle loss in disease. We finally explore the rationale of improving muscle regeneration by understanding and modulating fibro-adipogenic progenitors' fate and behavior.

In vitro assessment of anti-fibrotic drug activity does not predict in vivo efficacy in murine models of Duchenne muscular dystrophy.

AIM: Fibrosis is the most common complication from chronic diseases, and yet no therapy capable of mitigating its effects is available. Our goal is to unveil specific signaling regulating the fibrogenic process and to identify potential small molecule candidates that block fibrogenic differentiation of fibro/adipogenic progenitors. METHOD: We performed a large-scale drug screen using muscle-resident fibro/adipogenic progenitors from a mouse model expressing EGFP under the Collagen1a1 promotor. We first confirmed that the EGFP was expressed in response to TGFß1 stimulation in vitro. Then we treated cells with TGFß1 alone or with drugs from two libraries of known compounds. The drugs ability to block the fibrogenic differentiation was quantified by imaging and flow cytometry. From a two-rounds screening, positive hits were tested in vivo in the mice model for the Duchenne Muscular Dystrophy (mdx mice). The histopathology of the muscles was assessed with picrosirius red (fibrosis) and laminin staining (myofiber size). KEY FINDINGS: From the in vitro drug screening, we identified 21 drugs and tested 3 in vivo on the mdx mice. None of the three drugs significantly improved muscle histopathology. SIGNIFICANCE: The in vitro drug screen identified various efficient compounds, none of them strongly inhibited fibrosis in skeletal muscle of mdx mice. To explain these observations, we hypothesize that in Duchenne Muscular Dystrophy, in which fibrosis is a secondary event due to chronic degeneration and inflammation, the drugs tested could have adverse effect on regeneration or inflammation, balancing off any positive effects and leading to the absence of significant results.

TGF-ß-driven downregulation of the transcription factor TCF7L2 affects Wnt/ß-catenin signaling in PDGFRα+ fibroblasts.

Mesenchymal stromal cells (MSCs) are multipotent progenitors essential for organogenesis, tissue homeostasis, regeneration and scar formation. Tissue injury upregulates transforming growth factor ß (TGF-ß) signaling, which modulates myofibroblast fate, extracellular matrix remodeling and fibrosis. However, the molecular determinants of MSC differentiation and survival remain poorly understood. During canonical Wnt signaling, T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors regulate development and stemness, but the mechanisms by which injury-induced cues modulate their expression remain underexplored. Here, we studied the cell type-specific gene expression of TCF/LEF transcription factors and, more specifically, we investigated whether damage-induced TGF-ß signaling impairs the expression and function of TCF7L2 (also known as TCF4), using several models of MSCs, including skeletal muscle fibro-adipogenic progenitors. We show that TCF/LEFs are differentially expressed and that TGF-ß reduces the expression of TCF7L2 in MSCs but not in myoblasts. We also found that the ubiquitin-proteasome system regulates TCF7L2 proteostasis and participates in TGF-ß-mediated TCF7L2 protein downregulation. Finally, we show that TGF-ß requires histone deacetylase activity to repress the expression of TCF7L2. Thus, our work reports a novel interplay between TGF-ß and canonical Wnt signaling cascades in PDGFRα+ fibroblasts and suggests that this mechanism could be targeted in tissue repair and regeneration.

Single-cell revolution unveils the mysteries of the regenerative mammalian digit tip.

The digit tip is an exciting model for studying regeneration in mammals, but the precise mechanisms and the populations of cells involved in the formation and remodeling of the blastema remain unknown. In an exciting new work, Storer et al. take advantage of single-cell RNAseq combined with Pdgfra+ â€‹lineage-tracing to open the way into the enigmatic world of mammalian tissue regeneration.

Hic1 deletion unleashes quiescent connective tissue stem cells and impairs skeletal muscle regeneration.

Skeletal muscle fibro-adipogenic progenitors (FAPs) are tissue-resident connective tissue cells and the main cellular source of pathological fibro-fatty scar associated with muscle disorders. Although our knowledge about skeletal muscle mesenchymal progenitor cells has exploded in the past decade, we still lack information about their origin, fate, gene regulation, function, and stemness. A recent study by Underhill and colleagues, published in Cell Stem Cell, described the last census of Hic1 mesenchymal progenitor/stem cells in skeletal muscle regeneration, providing valuable results and data to the ever-expanding community of scientists interested in tissue regeneration and fibrosis. This commentary contextualizes and summarizes these exciting new findings.

Cross-talk between TGF-ß and PDGFRα signaling pathways regulates the fate of stromal fibro-adipogenic progenitors.

Fibro-adipogenic progenitors (FAPs) are tissue-resident mesenchymal stromal cells (MSCs) required for proper skeletal muscle development, regeneration and maintenance. However, FAPs are also responsible for fibro-fatty scar deposition following chronic damage. We aimed to investigate the role of functional cross-talk between TGF-ß and PDGFRα signaling pathways in the fate of FAPs. Here, we show that the number of FAPs correlates with TGF-ß levels and with extracellular matrix deposition during regeneration and repair. Interestingly, the expression of PDGFRα changed dynamically in the fibroblast lineage after injury. Furthermore, PDGFRα-dependent immediate early gene expression changed during regeneration and repair. We also found that TGF-ß signaling reduces PDGFRα expression in FAPs, mouse dermal fibroblasts and in two related mesenchymal cell lines. Moreover, TGF-ß promotes myofibroblast differentiation of FAPs but inhibits their adipogenicity. Accordingly, TGF-ß impairs the expression of PDGFRα-dependent immediate early genes in a TGFBR1-dependent manner. Finally, pharmacological inhibition of PDGFRα activity with AG1296 impaired TGF-ß-induced extracellular matrix remodeling, Smad2 signaling, myofibroblast differentiation and migration of MSCs. Thus, our work establishes a functional cross-talk between TGF-ß and PDGFRα signaling pathways that is involved in regulating the biology of FAPs and/or MSCs.This article has an associated First Person interview with the first author of the paper.

Denervation-induced skeletal muscle fibrosis is mediated by CTGF/CCN2 independently of TGF-ß.

Muscular fibrosis is caused by excessive accumulation of extracellular matrix (ECM) that replaces functional tissue, and it is a feature of several myopathies and neuropathies. Knowledge of the biology and regulation of pro-fibrotic factors is critical for the development of new therapeutic strategies. Upon unilateral sciatic nerve transection, we observed accumulation of ECM proteins such as collagen and fibronectin in the denervated hindlimb, together with increased levels of the profibrotic factors transforming growth factor type ß (TGF-ß) and connective tissue growth factor (CTGF/CCN2). In mice hemizygous for CTGF/CCN2 or in mice treated with a blocking antibody against CTGF/CCN2, we observed reduced accumulation of ECM proteins after denervation as compared to control mice, with no changes in fibro/adipogenic progenitors (FAPs), suggesting a direct role of CTGF/CCN2 on denervation-induced fibrosis. During time course experiments, we observed that ECM proteins and CTGF/CCN2 levels are increased early after denervation (2-4 days), while TGF-ß signaling shows a delayed kinetics of appearance (1-2 weeks). Furthermore, blockade of TGF-ß signaling does not decrease fibronectin or CTGF levels after 4 days of denervation. These results suggest that in our model CTGF/CCN2 is not up-regulated by canonical TGF-ß signaling early after denervation and that other factors are likely involved in the early fibrotic response following skeletal muscle denervation.

Adherent muscle connective tissue fibroblasts are phenotypically and biochemically equivalent to stromal fibro/adipogenic progenitors.

Extracellular matrix (ECM) gives structure, support, and is the niche for several cells found in skeletal muscle. ECM is mainly produced by muscle connective tissue (CT) fibroblasts during development and regeneration. Stromal fibroadipogenic progenitors (FAPs) are CT fibroblasts-like mesenchymal progenitors (MPs) with important roles in regeneration and degeneration. Chronic damage restrains the normal regenerative behavior of muscle fibroblasts/FAPs. Thus, the isolation and study of these mesenchymal progenitors are of crucial importance for understanding their behavior and biology. We investigated whether adult muscle CT fibroblasts (hereafter referred to as adherent fibroblasts [aFbs]) cultured via pre-plating strategy belong to a heterogeneous population of FAPs. By combining microscopy, western blot analyses, flow cytometry, and FACS we determined that aFbs isolated from skeletal muscle largely overlap with FAPs. In addition, we used the PDGFRαEGFP mice in order to corroborate our results with EGFP+ FAPs. Moreover, our strategy allows the isolation of activated EGFP+ FAPs from the murine DMD model PDGFRαEGFP; mdx and PDGFRαEGFP denervated mice. Here we report that 1 h 30 min of pre-plating strategy allows the isolation and culture of a highly enriched population of aFbs. These cells are phenotypically and biochemically a FAPs-like population of adherent cells. In addition, aFbs respond in the same fashion as FAPs to Nilotinib, an inducer of FAPs apoptosis. Moreover, flow cytometry characterization of these aFbs suggests that 85% of them express the MP marker PDGFRα, and isolation of aFbs from the PDGFRαEGFP mice suggests that 75% of them show high EGFP expression. Furthermore, TGF-ß1 induces aFbs proliferation, myofibroblast differentiation, and ECM production. We were also able to isolate activated aFbs from skeletal muscle of the DMD mice and from the PDGFRαEGFP mice 2-days after denervation. Our findings suggest that the in vitro pre-plating strategy allows the isolation and culture of a relatively pure aFbs population, which resembles FAPs in vitro.

Nilotinib impairs skeletal myogenesis by increasing myoblast proliferation.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are effective therapies with demonstrated antineoplastic activity. Nilotinib is a second-generation FDA-approved TKI designed to overcome Imatinib resistance and intolerance in patients with chronic myelogenous leukemia (CML). Interestingly, TKIs have also been shown to be an efficient treatment for several non-malignant disorders such fibrotic diseases, including those affecting skeletal muscles. METHODS: We investigated the role of Nilotinib on skeletal myogenesis using the well-established C2C12 myoblast cell line. We evaluated the impact of Nilotinib during the time course of skeletal myogenesis. We compared the effect of Nilotinib with the well-known p38 MAPK inhibitor SB203580. MEK1/2 UO126 and PI3K/AKT LY294002 inhibitors were used to identify the signaling pathways involved in Nilotinib-related effects on myoblast. Adult primary myoblasts were also used to corroborate the inhibition of myoblasts fusion and myotube-nuclei positioning by Nilotinib. RESULTS: We found that Nilotinib inhibited myogenic differentiation, reducing the number of myogenin-positive myoblasts and decreasing myogenin and MyoD expression. Furthermore, Nilotinib-mediated anti-myogenic effects impair myotube formation, myosin heavy chain expression, and compromise myotube-nuclei positioning. In addition, we found that p38 MAPK is a new off-target protein of Nilotinib, which causes inhibition of p38 phosphorylation in a similar manner as the well-characterized p38 inhibitor SB203580. Nilotinib induces the activation of ERK1/2 and AKT on myoblasts but not in myotubes. We also found that Nilotinib stimulates myoblast proliferation, a process dependent on ERK1/2 and AKT activation. CONCLUSIONS: Our findings suggest that Nilotinib may have important negative effects on muscle homeostasis, inhibiting myogenic differentiation but stimulating myoblasts proliferation. Additionally, we found that Nilotinib stimulates the activation of ERK1/2 and AKT. On the other hand, we suggest that p38 MAPK is a new off-target of Nilotinib. Thus, there is a necessity for future studies to investigate the long-term effects of TKIs on skeletal muscle homeostasis, along with potential detrimental effects in cell differentiation and proliferation in patients receiving TKI therapies.

Expression of CTGF/CCN2 in response to LPA is stimulated by fibrotic extracellular matrix via the integrin/FAK axis.

Fibrosis is a common feature of several chronic diseases and is characterized by exacerbated accumulation of ECM. An understanding of the cellular and molecular mechanisms involved in the development of this condition is crucial for designing efficient treatments for those pathologies. Connective tissue growth factor (CTGF/CCN2) is a pleiotropic protein with strong profibrotic activity. In this report, we present experimental evidence showing that ECM stimulates the synthesis of CTGF in response to lysophosphatidic acid (LPA).The integrin/focal adhesion kinase (FAK) signaling pathway mediates this effect, since CTGF expression is abolished by the use of the Arg-Gly-Asp-Ser peptide and also by an inhibitor of FAK autophosphorylation at tyrosine 397. Cilengitide, a specific inhibitor of αv integrins, inhibits the expression of CTGF mediated by LPA or transforming growth factor ß1. We show that ECM obtained from decellularized myofibroblast cultures or derived from activated fibroblasts from muscles of the Duchenne muscular dystrophy mouse model ( mdx) induces the expression of CTGF. This effect is dependent on FAK phosphorylation in response to its activation by integrin. We also found that the fibrotic ECM inhibits skeletal muscle differentiation. This novel regulatory mechanism of CTGF expression could be acting as a positive profibrotic feedback between the ECM and CTGF, revealing a novel concept in the control of fibrosis under chronic damage.

ALS skeletal muscle shows enhanced TGF-ß signaling, fibrosis and induction of fibro/adipogenic progenitor markers.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease in which upper and lower motoneurons degenerate leading to muscle wasting, paralysis and eventually death from respiratory failure. Several studies indicate that skeletal muscle contributes to disease progression; however the molecular mechanisms remain elusive. Fibrosis is a common feature in skeletal muscle under chronic damage conditions such as those caused by muscular dystrophies or denervation. However, the exact mechanisms of fibrosis induction and the cellular bases of this pathological response are unknown. We show that extracellular matrix (ECM) components are augmented in skeletal muscles of symptomatic hSOD1G93A mice, a widely used murine model of ALS. These mice also show increased TGF-ß1 mRNA levels, total Smad3 protein levels and p-Smad3 positive nuclei. Furthermore, platelet-derived growth factor receptor-α (PDGFRα), Tcf4 and α-smooth muscle actin (α-SMA) levels are augmented in the skeletal muscle of symptomatic hSOD1G93A mice. Additionally, the fibro/adipogenic progenitors (FAPs), which are the main producers of ECM constituents, are also increased in these pathogenic conditions. Therefore, FAPs and ECM components are more abundant in symptomatic stages of the disease than in pre-symptomatic stages. We present evidence that fibrosis observed in skeletal muscle of symptomatic hSOD1G93A mice is accompanied with an induction of TGF-ß signaling, and also that FAPs might be involved in triggering a fibrotic response. Co-localization of p-Smad3 positive cells together with PDGFRα was observed in the interstitial cells of skeletal muscles from symptomatic hSOD1G93A mice. Finally, the targeting of pro-fibrotic factors such as TGF-ß, CTGF/CCN2 and platelet-derived growth factor (PDGF) signaling pathway might be a suitable therapeutic approach to improve muscle function in several degenerative diseases.

Fibro/adipogenic progenitors safeguard themselves: a novel mechanism to reduce fibrosis is discovered.

PDGFRα regulates several cellular processes, and exacerbated PDGF signaling cause fibrosis in different tissues. Different research groups have shown that fibro/adipogenic progenitors (FAPs) are responsible for pathological fibrosis found in skeletal muscle disorders. Rando's Lab describes that an intronic polyadenylation of Pdgfra regulates FAPs activity, and therefore fibrosis. This discovery opens a new potential target for treating fibrosis.

Connective tissue cells expressing fibro/adipogenic progenitor markers increase under chronic damage: relevance in fibroblast-myofibroblast differentiation and skeletal muscle fibrosis.

Fibrosis occurs in skeletal muscle under various pathophysiological conditions such as Duchenne muscular dystrophy (DMD), a devastating disease characterized by fiber degeneration that results in progressive loss of muscle mass, weakness and increased extracellular matrix (ECM) accumulation. Fibrosis is also observed after skeletal muscle denervation and repeated cycles of damage followed by regeneration. The ECM is synthesized largely by fibroblasts in the muscle connective tissue under normal conditions. Myofibroblasts, cells that express α-smooth muscle actin (α-SMA), play a role in many tissues affected by fibrosis. In skeletal muscle, fibro/adipogenic progenitors (FAPs) that express cell-surface platelet-derived growth factor receptor-α (PDGFR-α) and the transcription factor Tcf4 seem to be responsible for connective tissue synthesis and are good candidates for the origin of myofibroblasts. We show that cells positive for Tcf4 and PDGFR-α are expressed in skeletal muscle under normal conditions and are increased in various skeletal muscles of mdx mice, a murine model for DMD, wild type muscle after sciatic denervation and muscle subjected to chronic damage. These cells co-label with the myofibroblast marker α-SMA in dystrophic muscle but not in normal tissue. The Tcf4-positive cells lie near macrophages mainly concentrated in dystrophic necrotic-regenerating foci. The close proximity of Tcf4-positive cells to inflammatory cells and their previously described role in muscle regeneration might reflect an active interaction between these cell types and growth factors, possibly resulting in a muscular regenerative or fibrotic condition.