The chemokine CCL1 triggers an AMFR-SPRY1 pathway that promotes differentiation of lung fibroblasts into myofibroblasts and drives pulmonary fibrosis.

Recruitment of immune cells to the site of inflammation by the chemokine CCL1 is important in the pathology of inflammatory diseases. Here, we examined the role of CCL1 in pulmonary fibrosis (PF). Bronchoalveolar lavage fluid from PF mouse models contained high amounts of CCL1, as did lung biopsies from PF patients. Immunofluorescence analyses revealed that alveolar macrophages and CD4+ T cells were major producers of CCL1 and targeted deletion of Ccl1 in these cells blunted pathology. Deletion of the CCL1 receptor Ccr8 in fibroblasts limited migration, but not activation, in response to CCL1. Mass spectrometry analyses of CCL1 complexes identified AMFR as a CCL1 receptor, and deletion of Amfr impaired fibroblast activation. Mechanistically, CCL1 binding triggered ubiquitination of the ERK inhibitor Spry1 by AMFR, thus activating Ras-mediated profibrotic protein synthesis. Antibody blockade of CCL1 ameliorated PF pathology, supporting the therapeutic potential of targeting this pathway for treating fibroproliferative lung diseases.

Identification of a myofibroblast differentiation program during neonatal lung development.

Alveologenesis is the final stage of lung development in which the internal surface area of the lung is increased to facilitate efficient gas exchange in the mature organism. The first phase of alveologenesis involves the formation of septal ridges (secondary septae) and the second phase involves thinning of the alveolar septa. Within secondary septa, mesenchymal cells include a transient population of alveolar myofibroblasts (MyoFBs) and a stable but poorly described population of lipid-rich cells that have been referred to as lipofibroblasts or matrix fibroblasts (MatFBs). Using a unique Fgf18CreER lineage trace mouse line, cell sorting, single-cell RNA sequencing and primary cell culture, we have identified multiple subtypes of mesenchymal cells in the neonatal lung, including an immature progenitor cell that gives rise to mature MyoFB. We also show that the endogenous and targeted ROSA26 locus serves as a sensitive reporter for MyoFB maturation. These studies identify a MyoFB differentiation program that is distinct from other mesenchymal cell types and increases the known repertoire of mesenchymal cell types in the neonatal lung.

Hippo signaling does it again: arbitrating cardiac fibroblast identity and activation.

The Hippo pathway is an evolutionarily conserved kinase cascade that is fundamental for tissue development, homeostasis, and regeneration. In the developing mammalian heart, Hippo signaling regulates cardiomyocyte numbers and organ size. While cardiomyocytes in the adult heart are largely postmitotic, Hippo deficiency can increase proliferation of these cells and affect cardiac regenerative capacity. Recent studies have also shown that resident cardiac fibroblasts play a critical role in disease responsiveness and healing, and in this issue of Genes and Development, Xiao and colleagues (pp. 1491-1505) demonstrate that Hippo signaling also integrates the activity of fibroblasts in the heart. They show that Hippo signaling normally maintains the cardiac fibroblast in a resting state and, conversely, its inactivation during disease-related stress results in a spontaneous transition toward a myofibroblast state that underlies fibrosis and ventricular remodeling. This phenotypic switch is associated with increased cytokine signaling that promotes nonautonomous resident fibroblast and myeloid cell activation.

Hippo pathway deletion in adult resting cardiac fibroblasts initiates a cell state transition with spontaneous and self-sustaining fibrosis.

Cardiac fibroblasts (CFs) respond to injury by transitioning through multiple cell states, including resting CFs, activated CFs, and myofibroblasts. We report here that Hippo signaling cell-autonomously regulates CF fate transitions and proliferation, and non-cell-autonomously regulates both myeloid and CF activation in the heart. Conditional deletion of Hippo pathway kinases, Lats1 and Lats2, in uninjured CFs initiated a self-perpetuating fibrotic response in the adult heart that was exacerbated by myocardial infarction (MI). Single cell transcriptomics showed that uninjured Lats1/2 mutant CFs spontaneously transitioned to a myofibroblast cell state. Through gene regulatory network reconstruction, we found that Hippo-deficient myofibroblasts deployed a network of transcriptional regulators of endoplasmic reticulum (ER) stress, and the unfolded protein response (UPR) consistent with elevated secretory activity. We observed an expansion of myeloid cell heterogeneity in uninjured Lats1/2 CKO hearts with similarity to cells recovered from control hearts post-MI. Integrated genome-wide analysis of Yap chromatin occupancy revealed that Yap directly activates myofibroblast cell identity genes, the proto-oncogene Myc, and an array of genes encoding pro-inflammatory factors through enhancer-promoter looping. Our data indicate that Lats1/2 maintain the resting CF cell state through restricting the Yap-induced injury response.

Sp3 is essential for normal lung morphogenesis and cell cycle progression during mouse embryonic development.

Members of the Sp family of transcription factors regulate gene expression via binding GC boxes within promoter regions. Unlike Sp1, which stimulates transcription, the closely related Sp3 can either repress or activate gene expression and is required for perinatal survival in mice. Here, we use RNA-seq and cellular phenotyping to show how Sp3 regulates murine fetal cell differentiation and proliferation. Homozygous Sp3-/- mice were smaller than wild-type and Sp+/- littermates, died soon after birth and had abnormal lung morphogenesis. RNA-seq of Sp3-/- fetal lung mesenchymal cells identified alterations in extracellular matrix production, developmental signaling pathways and myofibroblast/lipofibroblast differentiation. The lungs of Sp3-/- mice contained multiple structural defects, with abnormal endothelial cell morphology, lack of elastic fiber formation, and accumulation of lipid droplets within mesenchymal lipofibroblasts. Sp3-/- cells and mice also displayed cell cycle arrest, with accumulation in G0/G1 and reduced expression of numerous cell cycle regulators including Ccne1. These data detail the global impact of Sp3 on in vivo mouse gene expression and development.

A novel mechanoeffector role of fibroblast S100A4 in myofibroblast transdifferentiation and fibrosis.

Fibroblast to myofibroblast transdifferentiation mediates numerous fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fibrotic lung extracellular matrix, thereby mediating myofibroblast transdifferentiation. NMII-A is known to interact with the calcium-binding protein S100A4, but the mechanism by which S100A4 regulates fibrotic disorders is unclear. In this study, we show that fibroblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25 kPa) and thereby mediates myofibroblast transdifferentiation. Re-expression of endogenous fibroblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fibroblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine in vivo pulmonary fibrosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fibroblast S100A4 in inducing cytoskeletal redistribution in fibrotic disorders such as IPF.

Single-cell RNA sequencing reveals PDGFRα+ stromal cell subpopulations that promote proacinar cell differentiation in embryonic salivary gland organoids.

Stromal cells can direct the differentiation of epithelial progenitor cells during organ development. Fibroblast growth factor (FGF) signaling is essential for submandibular salivary gland development. Through stromal fibroblast cells, FGF2 can indirectly regulate proacinar cell differentiation in organoids, but the mechanisms are not understood. We performed single-cell RNA-sequencing and identified multiple stromal cell subsets, including Pdgfra+ stromal subsets expressing both Fgf2 and Fgf10. When combined with epithelial progenitor cells in organoids, magnetic-activated cell-sorted PDGFRα+ cells promoted proacinar cell differentiation similarly to total stroma. Gene expression analysis revealed that FGF2 increased the expression of multiple stromal genes, including Bmp2 and Bmp7. Both BMP2 and BMP7 synergized with FGF2, stimulating proacinar cell differentiation but not branching. However, stromal cells grown without FGF2 did not support proacinar organoid differentiation and instead differentiated into myofibroblasts. In organoids, TGFß1 treatment stimulated myofibroblast differentiation and inhibited the proacinar cell differentiation of epithelial progenitor cells. Conversely, FGF2 reversed the effects of TGFß1. We also demonstrated that adult salivary stromal cells were FGF2 responsive and could promote proacinar cell differentiation. These FGF2 signaling pathways may have applications in future regenerative therapies.

Procyanidin C1 inhibits bleomycin-induced pulmonary fibrosis in mice by selective clearance of senescent myofibroblasts.

Pulmonary fibrosis is a formidable challenge in chronic and age-related lung diseases. Myofibroblasts secrete large amounts of extracellular matrix and induce pro-repair responses during normal wound healing. Successful tissue repair results in termination of myofibroblast activity via apoptosis; however, some myofibroblasts exhibit a senescent phenotype and escape apoptosis, causing over-repair that is characterized by pathological fibrotic scarring. Therefore, the removal of senescent myofibroblasts using senolytics is an important method for the treatment of pulmonary fibrosis. Procyanidin C1 (PCC1) has recently been discovered as a senolytic compound with very low toxicity and few side effects. This study aimed to determine whether PCC1 could improve lung fibrosis by promoting apoptosis in senescent myofibroblasts and to investigate the mechanisms involved. The results showed that PCC1 attenuates bleomycin (BLM)-induced pulmonary fibrosis in mice. In addition, we found that PCC1 inhibited extracellular matrix deposition and promoted the apoptosis of senescent myofibroblasts by increasing PUMA expression and activating the BAX signaling pathway. Our findings represent a new method of pulmonary fibrosis management and emphasize the potential of PCC1 as a senotherapeutic agent for the treatment of pulmonary fibrosis, providing hope for patients with pulmonary fibrosis worldwide. Our results advance our understanding of age-related diseases and highlight the importance of addressing cellular senescence in treatment.

Dexamethasone induces trabecular meshwork cell myofibroblast transdifferentiation through ARHGEF26.

Glucocorticoid use may cause elevated intraocular pressure, leading to the development of glucocorticoid-induced glaucoma (GIG). However, the mechanism of GIG development remains incompletely understood. In this study, we subjected primary human trabecular meshwork cells (TMCs) and mice to dexamethasone treatment to mimic glucocorticoid exposure. The myofibroblast transdifferentiation of TMCs was observed in cellular and mouse models, as well as in human trabecular mesh specimens. This was demonstrated by the cytoskeletal reorganization, alterations in cell morphology, heightened transdifferentiation markers, increased extracellular matrix deposition, and cellular dysfunction. Knockdown of Rho guanine nucleotide exchange factor 26 (ARHGEF26) expression ameliorated dexamethasone-induced changes in cell morphology and upregulation of myofibroblast markers, reversed dysfunction and extracellular matrix deposition in TMCs, and prevented the development of dexamethasone-induced intraocular hypertension. And, this process may be related to the TGF-ß pathway. In conclusion, glucocorticoids induced the myofibroblast transdifferentiation in TMCs, which played a crucial role in the pathogenesis of GIG. Inhibition of ARHGEF26 expression protected TMCs by reversing myofibroblast transdifferentiation. This study demonstrated the potential of reversing the myofibroblast transdifferentiation of TMCs as a new target for treating GIG.

Multi-organ single-cell RNA sequencing in mice reveals early hyperglycemia responses that converge on fibroblast dysregulation.

Diabetes causes a range of complications that can affect multiple organs. Hyperglycemia is an important driver of diabetes-associated complications, mediated by biological processes such as dysfunction of endothelial cells, fibrosis, and alterations in leukocyte number and function. Here, we dissected the transcriptional response of key cell types to hyperglycemia across multiple tissues using single-cell RNA sequencing (scRNA-seq) and identified conserved, as well as organ-specific, changes associated with diabetes complications. By studying an early time point of diabetes, we focus on biological processes involved in the initiation of the disease, before the later organ-specific manifestations had supervened. We used a mouse model of type 1 diabetes and performed scRNA-seq on cells isolated from the heart, kidney, liver, and spleen of streptozotocin-treated and control male mice after 8 weeks and assessed differences in cell abundance, gene expression, pathway activation, and cell signaling across organs and within organs. In response to hyperglycemia, endothelial cells, macrophages, and monocytes displayed organ-specific transcriptional responses, whereas fibroblasts showed similar responses across organs, exhibiting altered metabolic gene expression and increased myeloid-like fibroblasts. Furthermore, we found evidence of endothelial dysfunction in the kidney, and of endothelial-to-mesenchymal transition in streptozotocin-treated mouse organs. In summary, our study represents the first single-cell and multi-organ analysis of early dysfunction in type 1 diabetes-associated hyperglycemia, and our large-scale dataset (comprising 67 611 cells) will serve as a starting point, reference atlas, and resource for further investigating the events leading to early diabetic disease.

Opposing roles for ADAMTS2 and ADAMTS14 in myofibroblast differentiation and function.

Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor ß availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Increased extracellular matrix stiffness regulates myofibroblast transformation through induction of autophagy-mediated Kindlin-2 cytoplasmic translocation.

The extracellular matrix (ECM) mechanical properties regulate biological processes, such as fibroblast-myofibroblast transformation (FMT), which is a crucial component in pelvic organ prolapse (POP) development. The 'Kindlin-2' protein, expressed by fibroblasts, plays an important role in the development of the mesoderm, which is responsible for connective tissue formation; however, the role of Kindlin-2 in FMT remains to be explored. In this study, we aimed to explore the role of Kindlin-2 in FMT as it relates to POP. We found that ECM stiffness induces autophagy to translocate Kindlin-2 to the cytoplasm of L929 cells, where it interacts with and degrades MOB1, thereby facilitating Yes-associated protein (YAP) entry into the nucleus and influencing FMT progression. Stiffness-induced autophagy was inhibited when using an autophagy inhibitor, which blocked the translocation of Kindlin-2 to the cytoplasm and partially reversed high-stiffness-induced FMT. In patients with POP, we observed an increase in cytoplasmic Kindlin-2 and nuclear YAP levels. Similar changes in vaginal wall-associated proteins were observed in a mouse model of acute vaginal injury. In conclusion, Kindlin-2 is a key gene affecting ECM stiffness, which regulates FMT by inducing autophagy and may influence the development of POP.

Tetrahedral DNA loaded siCCR2 restrains M1 macrophage polarization to ameliorate pulmonary fibrosis in chemoradiation-induced murine model.

Idiopathic pulmonary fibrosis (IPF) is a chronic lethal disease in the absence of demonstrated efficacy for preventing progression. Although macrophage-mediated alveolitis is determined to participate in myofibrotic transition during disease development, the paradigm of continuous macrophage polarization is still under-explored due to lack of proper animal models. Here, by integrating 2.5 U/kg intratracheal Bleomycin administration and 10 Gy thorax irradiation at day 7, we generated a murine model with continuous alveolitis-mediated fibrosis, which mimics most of the clinical features of our involved IPF patients. In combination with data from scRNA-seq of patients and a murine IPF model, a decisive role of CCL2/CCR2 axis in driving M1 macrophage polarization was revealed, and M1 macrophage was further confirmed to boost alveolitis in leading myofibroblast activation. Multiple sticky-end tetrahedral framework nucleic acids conjunct with quadruple ccr2-siRNA (FNA-siCCR2) was synthesized in targeting M1 macrophages. FNA-siCCR2 successfully blocked macrophage accumulation in pulmonary parenchyma of the IPF murine model, thus preventing myofibroblast activation and leading to the disease remitting. Overall, our studies lay the groundwork to develop a novel IPF murine model, reveal M1 macrophages as potential therapeutic targets, and establish new treatment strategy by using FNA-siCCR2, which are highly relevant to clinical scenarios and translational research in the field of IPF.

Network modeling predicts personalized gene expression and drug responses in valve myofibroblasts cultured with patient sera.

Aortic valve stenosis (AVS) patients experience pathogenic valve leaflet stiffening due to excessive extracellular matrix (ECM) remodeling. Numerous microenvironmental cues influence pathogenic expression of ECM remodeling genes in tissue-resident valvular myofibroblasts, and the regulation of complex myofibroblast signaling networks depends on patient-specific extracellular factors. Here, we combined a manually curated myofibroblast signaling network with a data-driven transcription factor network to predict patient-specific myofibroblast gene expression signatures and drug responses. Using transcriptomic data from myofibroblasts cultured with AVS patient sera, we produced a large-scale, logic-gated differential equation model in which 11 biochemical and biomechanical signals were transduced via a network of 334 signaling and transcription reactions to accurately predict the expression of 27 fibrosis-related genes. Correlations were found between personalized model-predicted gene expression and AVS patient echocardiography data, suggesting links between fibrosis-related signaling and patient-specific AVS severity. Further, global network perturbation analyses revealed signaling molecules with the most influence over network-wide activity, including endothelin 1 (ET1), interleukin 6 (IL6), and transforming growth factor ß (TGFß), along with downstream mediators c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription (STAT), and reactive oxygen species (ROS). Lastly, we performed virtual drug screening to identify patient-specific drug responses, which were experimentally validated via fibrotic gene expression measurements in valvular interstitial cells cultured with AVS patient sera and treated with or without bosentan-a clinically approved ET1 receptor inhibitor. In sum, our work advances the ability of computational approaches to provide a mechanistic basis for clinical decisions including patient stratification and personalized drug screening.

Fibroblast heterogeneity and tertiary lymphoid tissues in the kidney.

Fibroblasts reside in various organs and support tissue structure and homeostasis under physiological conditions. Phenotypic alterations of fibroblasts underlie the development of diverse pathological conditions, including organ fibrosis. Recent advances in single-cell biology have revealed that fibroblasts comprise heterogeneous subpopulations with distinct phenotypes, which exert both beneficial and detrimental effects on the host organs in a context-dependent manner. In the kidney, phenotypic alterations of resident fibroblasts provoke common pathological conditions of chronic kidney disease (CKD), such as renal anemia and peritubular capillary loss. Additionally, in aged injured kidneys, fibroblasts provide functional and structural supports for tertiary lymphoid tissues (TLTs), which serve as the ectopic site of acquired immune reactions in various clinical contexts. TLTs are closely associated with aging and CKD progression, and the developmental stages of TLTs reflect the severity of renal injury. In this review, we describe the current understanding of fibroblast heterogeneity both under physiological and pathological conditions, with special emphasis on fibroblast contribution to TLT formation in the kidney. Dissecting the heterogeneous characteristics of fibroblasts will provide a promising therapeutic option for fibroblast-related pathological conditions, including TLT formation.

The inflammatory speech of fibroblasts.

Activation of fibroblasts is a key event during normal tissue repair after injury and the dysregulated repair processes that result in organ fibrosis. To most researchers, fibroblasts are rather unremarkable spindle-shaped cells embedded in the fibrous collagen matrix of connective tissues and/or deemed useful to perform mechanistic studies with adherent cells in culture. For more than a century, fibroblasts escaped thorough classification due to the lack of specific markers and were treated as the leftovers after all other cells have been identified from a tissue sample. With novel cell lineage tracing and single cell transcriptomics tools, bona fide fibroblasts emerge as only one heterogeneous sub-population of a much larger group of partly overlapping cell types, including mesenchymal stromal cells, fibro-adipogenic progenitor cells, pericytes, and/or perivascular cells. All these cells are activated to contribute to tissue repair after injury and/or chronic inflammation. "Activation" can entail various functions, such as enhanced proliferation, migration, instruction of inflammatory cells, secretion of extracellular matrix proteins and organizing enzymes, and acquisition of a contractile myofibroblast phenotype. We provide our view on the fibroblastic cell types and activation states playing a role during physiological and pathological repair and their crosstalk with inflammatory macrophages. Inflammation and fibrosis of the articular synovium during rheumatoid arthritis and osteoarthritis are used as specific examples to discuss inflammatory fibroblast phenotypes. Ultimately, delineating the precursors and functional roles of activated fibroblastic cells will contribute to better and more specific intervention strategies to treat fibroproliferative and fibrocontractive disorders.

Drebrin is Required for Myosin-facilitated Actin Cytoskeletal Remodeling during Pulmonary Alveolar Development.

Alveolar septation increases gas-exchange surface area and requires coordinated cytoskeletal rearrangement in lung fibroblasts (LFs) to balance the demands of contraction and cell migration. We hypothesized that DBN (drebrin), a modulator of the actin cytoskeleton in neuronal dendrites, regulates the remodeling of the LF cytoskeleton. Using mice bearing a transgelin-Cre-targeted deletion of Dbn in pulmonary fibroblasts and pericytes, we examined alterations in alveolar septal outgrowth, LF spreading and migration, and actomyosin function. The alveolar surface area and number of alveoli were reduced, whereas alveolar ducts were enlarged, in mice bearing the dbn deletion (DBNΔ) compared with their littermates bearing only one dbn-Flox allele (control). Cultured DBNΔ LFs were deficient in their responses to substrate rigidity and migrated more slowly. Drebrin was abundant in the actin cortex and lamella, and the actin fiber orientation was less uniform in lamella of DBNΔ LFs, which limited the development of traction forces and altered focal adhesion dynamics. Actin fiber orientation is regulated by contractile NM2 (nonmuscle myosin-2) motors, which help arrange actin stress fibers into thick ventral actin stress fibers. Using fluorescence anisotropy, we observed regional intracellular differences in myosin regulatory light chain phosphorylation in control LFs that were altered by dbn deletion. Using perturbations to induce and then release stalling of NM2 on actin in LFs from both genotypes, we made predictions explaining how DBN interacts with actin and NM2. These studies provide new insight for diseases such as emphysema and pulmonary fibrosis, in which fibroblasts inappropriately respond to mechanical cues in their environment.

Tenascin-C in fibrosis in multiple organs: Translational implications.

Systemic sclerosis (SSc, scleroderma) is a complex disease with a pathogenic triad of autoimmunity, vasculopathy, and fibrosis involving the skin and multiple internal organs [1]. Because fibrosis accounts for as much as 45% of all deaths worldwide and appears to be increasing in prevalence [2], understanding its pathogenesis and progression is an urgent scientific challenge. Fibroblasts and myofibroblasts are the key effector cells executing physiologic tissue repair on one hand, and pathological fibrogenesis leading to chronic fibrosing conditions on the other. Recent studies identify innate immune signaling via toll-like receptors (TLRs) as a key driver of persistent fibrotic response in SSc. Repeated injury triggers the in-situ generation of "damage-associated molecular patterns" (DAMPs) or danger signals. Sensing of these danger signals by TLR4 on resident cells elicits potent stimulatory effects on fibrotic gene expression and myofibroblast differentiation triggering the self-limited tissue repair response to self-sustained pathological fibrosis characteristic of SSc. Our unbiased survey for DAMPs associated with SSc identified extracellular matrix glycoprotein tenascin-C as one of the most highly up-regulated ECM proteins in SSc skin and lung biopsies [3,4]. Furthermore, tenascin C is responsible for driving sustained fibroblasts activation, thereby progression of fibrosis [3]. This review summarizes recent studies examining the regulation and complex functional role of tenascin C, presenting tenascin-TLR4 axis in pathological fibrosis, and novel anti-fibrotic approaches targeting their signaling.

The well-developed actin cytoskeleton and Cthrc1 expression by actin-binding protein drebrin in myofibroblasts promote cardiac and hepatic fibrosis.

Fibrosis is mainly triggered by inflammation in various tissues, such as heart and liver tissues, and eventually leads to their subsequent dysfunction. Fibrosis is characterized by the excessive accumulation of extracellular matrix proteins (e.g., collagens) produced by myofibroblasts. The well-developed actin cytoskeleton of myofibroblasts, one of the main features differentiating them from resident fibroblasts in tissues under inflammatory conditions, contributes to maintaining their ability to produce excessive extracellular matrix proteins. However, the molecular mechanisms via which the actin cytoskeleton promotes the production of fibrosis-related genes in myofibroblasts remain unclear. In this study, we found, via single-cell analysis, that developmentally regulated brain protein (drebrin), an actin-binding protein, was specifically expressed in cardiac myofibroblasts with a well-developed actin cytoskeleton in fibrotic hearts. Moreover, our immunocytochemistry analysis revealed that drebrin promoted actin cytoskeleton formation and myocardin-related transcription factor-serum response factor signaling. Comprehensive single-cell analysis and RNA-Seq revealed that the expression of collagen triple helix repeat containing 1 (Cthrc1), a fibrosis-promoting secreted protein, was regulated by drebrin in cardiac myofibroblasts via myocardin-related transcription factor-serum response factor signaling. Furthermore, we observed the profibrotic effects of drebrin exerted via actin cytoskeleton formation and the Cthrc1 expression regulation by drebrin in liver myofibroblasts (hepatic stellate cells). Importantly, RNA-Seq demonstrated that drebrin expression levels increased in human fibrotic heart and liver tissues. In summary, our results indicated that the well-developed actin cytoskeleton and Cthrc1 expression due to drebrin in myofibroblasts promoted cardiac and hepatic fibrosis, suggesting that drebrin is a therapeutic target molecule for fibrosis.

AdipoRon reduces TGFß1-mediated collagen deposition in vitro and alleviates knee stiffness in vivo.

Arthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti-fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFß1-mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFß1-induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA-seq analysis corroborated the anti-myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle-treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis.