Dynamic Hippo pathway activity underlies mesenchymal differentiation during lung alveolar morphogenesis.

Alveologenesis, the final stage in lung development, substantially remodels the distal lung, expanding the alveolar surface area for efficient gas exchange. Secondary crest myofibroblasts (SCMF) exist transiently in the neonatal distal lung and are crucial for alveologenesis. However, the pathways that regulate SCMF function, proliferation and temporal identity remain poorly understood. To address this, we purified SCMFs from reporter mice, performed bulk RNA-seq and found dynamic changes in Hippo-signaling components during alveologenesis. We deleted the Hippo effectors Yap/Taz from Acta2-expressing cells at the onset of alveologenesis, causing a significant arrest in alveolar development. Using single cell RNA-seq, we identified a distinct cluster of cells in mutant lungs with altered expression of marker genes associated with proximal mesenchymal cell types, airway smooth muscle and alveolar duct myofibroblasts. In vitro studies confirmed that Yap/Taz regulates myofibroblast-associated gene signature and contractility. Together, our findings show that Yap/Taz is essential for maintaining functional myofibroblast identity during postnatal alveologenesis.

CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

Integration of transcriptomics and metabolomics reveals pathways involved in MDSC supernatant attenuation of TGF-ß1-induced myofibroblastic differentiation of mesenchymal stem cells.

Overexposure to transforming growth factor b1 (TGF-ß1) induces myofibroblastic differentiation of mesenchymal stem cells (MSCs), which could be attenuated by myeloid-derived suppressor cell (MDSC) supernatant. However, the promyofibroblastic effects of TGF-ß1 and the antimyofibroblastic effects of MDSC supernatant in MSCs have not been fully elucidated. To further clarify the latent mechanism and identify underlying therapeutic targets, we used an integrative strategy combining transcriptomics and metabolomics. Bone marrow MSCs were collected 24 h following TGF-ß1 and MDSC supernatant treatment for RNA sequencing and untargeted metabolomic analysis. The integrated data were then analyzed to identify significant gene-metabolite correlations. Differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) were assessed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for exploring the mechanisms of myofibroblastic differentiation of MSCs. The integration of transcriptomic and metabolomic data highlighted significantly coordinated changes in glycolysis/gluconeogenesis and purine metabolism following TGF-ß1 and MDSC supernatant treatment. By combining transcriptomic and metabolomic analyses, this study showed that glycolysis/gluconeogenesis and purine metabolism were essential for the myofibroblastic differentiation of MSCs and may serve as promising targets for mechanistic research and clinical practice in the treatment of fibrosis by MDSC supernatant.

CXCL4 drives fibrosis by promoting several key cellular and molecular processes.

Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.

Histamine Deficiency Promotes Myofibroblasts Transformation from HDC-Expressing CD11b+ Myeloid Cells in Injured Hearts Post Myocardial Infarction.

Myocardial infarction (MI) is a significant contributor to the development of heart failure. Histidine decarboxylase (HDC), the unique enzyme that converts L-histidine to histamine, is highly expressed in CD11b+ immature myeloid cells. However, the relationship between HDC-expressing macrophages and cardiac myofibroblasts remains to be explained. Here, we demonstrate that the GFP (green fluorescent protein)-labeled HDC+CD11b+ myeloid precursors and their descendants could differentiate into fibroblast-like cells in myocardial interstitium. Furthermore, we prove that CD11b+Ly6C+ monocytes/macrophages, but not CD11b+Ly6G+ granulocytes, are identified as the main cellular source for bone marrow-derived myofibroblast transformation, which could be regulated via histamine H1 and H2 receptor-dependent signaling pathways. Using HDC knockout mice, we find that histamine deficiency promotes myofibroblast transformation from Ly6C+ macrophages and cardiac fibrosis partly through upregulating the expression of Krüppel-like factor 5 (KLF5). Taken together, our data uncover a central role of HDC in regulating bone marrow-derived macrophage-to-myofibroblast transformation but also identify a histamine receptor (HR)-KLF5 related signaling pathway that mediates myocardial fibrosis post-MI. CD11b+Ly6C+ monocytes/macrophages are the main cellular source for bone marrow-derived myofibroblast transformation. Histamine inhibits myofibroblasts transformation via H1R and H2R-dependent signaling pathways, and ameliorates cardiac fibrosis partly through upregulating KLF5 expression.

Dexmedetomidine Promotes Lipopolysaccharide-Induced Differentiation of Cardiac Fibroblasts and Collagen I/III Synthesis through α2A Adrenoreceptor-Mediated Activation of the PKC-p38-Smad2/3 Signaling Pathway in Mice.

Dexmedetomidine (DEX), a selective α2 adrenergic receptor (AR) agonist, is commonly used as a sedative drug during critical illness. In the present study, we explored a novel accelerative effect of DEX on cardiac fibroblast (CF) differentiation mediated by LPS and clarified its potential mechanism. LPS apparently increased the expression of α-SMA and collagen I/III and the phosphorylation of p38 and Smad-3 in the CFs of mice. These effects were significantly enhanced by DEX through increasing α2A-AR expression in CFs after LPS stimulation. The CFs from α2A-AR knockout mice were markedly less sensitive to DEX treatment than those of wild-type mice. Inhibition of protein kinase C (PKC) abolished the enhanced effects of DEX on LPS-induced differentiation of CFs. We also found that the α-SMA level in the second-passage CFs was much higher than that in the nonpassage and first-passage CFs. However, after LPS stimulation, the TNF-α released from the nonpassage CFs was much higher than that in the first- and second-passage CFs. DEX had no effect on LPS-induced release of TNF-α and IL-6 from CFs. Further investigation indicated that DEX promoted cardiac fibrosis and collagen I/III synthesis in mice exposed to LPS for four weeks. Our results demonstrated that DEX effectively accelerated LPS-induced differentiation of CFs to myofibroblasts through the PKC-p38-Smad2/3 signaling pathway by activating α2A-AR.

Fibroblast to myofibroblast transition is enhanced by increased cell density.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung caused by a rampant inflammatory response that results in the deposition of excessive extracellular matrix (ECM). IPF patient lungs also develop fibroblastic foci that consist of activated fibroblasts and myofibroblasts. In concert with ECM deposition, the increased cell density within fibroblastic foci imposes confining forces on lung fibroblasts. In this work, we observed that increased cell density increases the incidence of the fibroblast-to-myofibroblast transition (FMT), but mechanical confinement imposed by micropillars has no effect on FMT incidence. We found that human lung fibroblasts (HLFs) express more α-SMA and deposit more collagen matrix, which are both characteristics of myofibroblasts, in response to TGF-ß1 when cells are seeded at a high density compared with a medium or a low density. These results support the hypothesis that HLFs undergo FMT more readily in response to TGF-ß1 when cells are densely packed, and this effect could be dependent on increased OB-cadherin expression. This work demonstrates that cell density is an important factor to consider when modelling IPF in vitro, and it may suggest decreasing cell density within fibroblastic foci as a strategy to reduce IPF burden.

Myofibroblast transdifferentiation is associated with changes in cellular and extracellular vesicle miRNA abundance.

Transforming growth factor-beta 1 (TGF-ß1), a pro-fibrotic tumour-derived factor promotes fibroblast differentiation in the tumour microenvironment and is thought to contribute to the development of pro-tumourigenic cancer-associated fibroblasts (CAFs) by promoting myofibroblast differentiation. miRNA dysregulation has been demonstrated in myofibroblast transdifferentiation and CAF activation, however, their expression varies among cell types and with the method of fibroblast induction. Here, the expression profile of miRNA in human primary oral fibroblasts treated with TGF-ß1, to derive a myofibroblastic, CAF-like phenotype, was determined compared to untreated fibroblasts. Myofibroblast transdifferentiation was determined by the expression of alpha-smooth muscle actin (α-SMA) and fibronectin-1 extra domain A (FN-EDA1) using quantitative real-time PCR (qRT-PCR) and western blot. The formation of stress fibres was assessed by fluorescence microscopy, and associated changes in contractility were assessed using collagen contraction assays. Extracellular vesicles (EVs) were purified by using size exclusion chromatography and ultracentrifugation and their size and concentration were determined by nanoparticle tracking analysis. miRNA expression profiling in oral fibroblasts treated with TGF-ß1 and their extracellular vesicles was carried out using tiling low-density array cards. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform functional and pathway enrichment analysis of target genes. In this study, TGF-ß1 induced a myofibroblastic phenotype in normal oral fibroblasts as assessed by expression of molecular markers, the formation of stress fibres and increased contractility. TaqMan Low-Density Array (TLDA) analysis demonstrated that miR-503 and miR-708 were significantly upregulated, while miR-1276 was significantly downregulated in TGF-ß1-treated oral fibroblasts (henceforth termed experimentally-derived CAF, eCAF). The gene functional enrichment analysis showed that the candidate miRNAs have the potential to modulate various pathways; including the Ras associated protein 1 (Rap1), PI3K-Akt, and tumour necrosis factor (TNF) signalling pathways. In addition, altered levels of several miRNAs were detected in eCAF EV, including miR-142 and miR-222. No differences in size or abundance of EV were detected between eCAF and normal oral fibroblast (NOF). Little overlap was observed between changes in cellular and EV miRNA profiles, suggesting the possibility of selective loading of EV miRNA. The study reveals miRNA expression signature could be involved in myofibroblast transdifferentiation and the miRNA cargo of their EV, providing novel insight into the involvement of miRNA in CAF development and function.

Effect of Pirfenidone on TGF-ß1-Induced Myofibroblast Differentiation and Extracellular Matrix Homeostasis of Human Orbital Fibroblasts in Graves' Ophthalmopathy.

Pirfenidone is a pyridinone derivative that has been shown to inhibit fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Its effect on orbital fibroblasts remains poorly understood. We investigated the in vitro effect of pirfenidone in transforming growth factor-ß1 (TGF-ß1)-induced myofibroblast transdifferentiation and extracellular matrix (ECM) homeostasis in primary cultured orbital fibroblasts from patients with Graves' ophthalmopathy (GO). The expression of fibrotic proteins, including α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), fibronectin, and collagen type I, was determined by Western blots. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for the ECM homeostasis were examined. After pretreating the GO orbital fibroblasts with pirfenidone (250, 500, and 750 µg/mL, respectively) for one hour followed by TGF-ß1 for another 24 h, the expression of α-SMA, CTGF, fibronectin, and collagen type I decreased in a dose-dependent manner. Pretreating the GO orbital fibroblasts with pirfenidone not only abolished TGF-ß1-induced TIMP-1 expression but recovered the MMP-2/-9 activities. Notably, pirfenidone inhibited TGF-ß1-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK), the critical mediators in the TGF-ß1 pathways. These findings suggest that pirfenidone modulates TGF-ß1-mediated myofibroblast differentiation and ECM homeostasis by attenuating downstream signaling of TGF-ß1.

Partial Ablation of Non-Myogenic Progenitor Cells as a Therapeutic Approach to Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), caused by the loss of dystrophin, remains incurable. Reduction in muscle regeneration with DMD is associated with the accumulation of fibroadipogenic progenitors (FAPs) differentiating into myofibroblasts and leading to a buildup of the collagenous tissue aggravating DMD pathogenesis. Mesenchymal stromal cells (MSCs) expressing platelet-derived growth factor receptors (PDGFRs) are activated in muscle during DMD progression and give rise to FAPs promoting DMD progression. Here, we hypothesized that muscle dysfunction in DMD could be delayed via genetic or pharmacologic depletion of MSC-derived FAPs. In this paper, we test this hypothesis in dystrophin-deficient mdx mice. To reduce fibro/adipose infiltration and potentiate muscle progenitor cells (MPCs), we used a model for inducible genetic ablation of proliferating MSCs via a suicide transgene, viral thymidine kinase (TK), expressed under the Pdgfrb promoter. We also tested if MSCs from fat tissue, the adipose stromal cells (ASCs), contribute to FAPs and could be targeted in DMD. Pharmacological ablation was performed with a hunter-killer peptide D-CAN targeting ASCs. MSC depletion with these approaches resulted in increased endurance, measured based on treadmill running, as well as grip strength, without significantly affecting fibrosis. Although more research is needed, our results suggest that depletion of pathogenic MSCs mitigates muscle damage and delays the loss of muscle function in mouse models of DMD.

Osthole Attenuates Bleomycin-Induced Pulmonary Fibrosis by Modulating NADPH Oxidase 4-Derived Oxidative Stress in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease characterized by the extensive accumulation of myofibroblasts and collagens. However, the exact mechanism that underlies this condition is unclear. Growing evidence suggests that NADPH oxidases (NOXs), especially NOX4-derived oxidative stress, play an important role in the development of lung fibrosis. Bleomycin (BLM) is a tumor chemotherapeutic agent, which has been widely employed to establish IPF animal models. Osthole (OST) is an active constituent of the fruit of Cnidium ninidium. Here, we used an in vivo mouse model and found that OST suppressed BLM-induced body weight loss, lung injury, pulmonary index increase, fibroblast differentiation, and pulmonary fibrosis. OST also significantly downregulated BLM-induced NOX4 expression and oxidative stress in the lungs. In vitro, OST could inhibit TGF-ß1-induced Smad3 phosphorylation, differentiation, proliferation, collagen synthesis, NOX4 expression, and ROS generation in human lung fibroblasts in a concentration-dependent manner. Moreover, NOX4 overexpression could prevent the above effects of OST. We came to the conclusion that OST could significantly attenuate BLM-induced pulmonary fibrosis in mice, via the mechanism that involved downregulating TGF-ß1/NOX4-mediated oxidative stress in lung fibroblasts.

PRMT7 targets of Foxm1 controls alveolar myofibroblast proliferation and differentiation during alveologenesis.

Although aberrant alveolar myofibroblasts (AMYFs) proliferation and differentiation are often associated with abnormal lung development and diseases, such as bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF), epigenetic mechanisms regulating proliferation and differentiation of AMYFs remain poorly understood. Protein arginine methyltransferase 7 (PRMT7) is the only reported type III enzyme responsible for monomethylation of arginine residue on both histone and nonhistone substrates. Here we provide evidence for PRMT7's function in regulating AMYFs proliferation and differentiation during lung alveologenesis. In PRMT7-deficient mice, we found reduced AMYFs proliferation and differentiation, abnormal elastin deposition, and failure of alveolar septum formation. We further shown that oncogene forkhead box M1 (Foxm1) is a direct target of PRMT7 and that PRMT7-catalyzed monomethylation at histone H4 arginine 3 (H4R3me1) directly associate with chromatin of Foxm1 to activate its transcription, and thereby regulate of cell cycle-related genes to inhibit AMYFs proliferation and differentiation. Overexpression of Foxm1 in isolated myofibroblasts (MYFs) significantly rescued PRMT7-deficiency-induced cell proliferation and differentiation defects. Thus, our results reveal a novel epigenetic mechanism through which PRMT7-mediated histone arginine monomethylation activates Foxm1 transcriptional expression to regulate AMYFs proliferation and differentiation during lung alveologenesis and may represent a potential target for intervention in pulmonary diseases.

GED-0507 attenuates lung fibrosis by counteracting myofibroblast transdifferentiation in vivo and in vitro.

The development of more effective, better tolerated drug treatments for progressive pulmonary fibrosis (of which idiopathic pulmonary fibrosis is the most common and severe form) is a research priority. The peroxisome proliferator-activated receptor gamma (PPAR-γ) is a key regulator of inflammation and fibrosis and therefore represents a potential therapeutic target. However, the use of synthetic PPAR-γ agonists may be limited by their potentially severe adverse effects. In a mouse model of bleomycin (BLM)-induced pulmonary fibrosis, we have demonstrated that the non-racemic selective PPAR-γ modulator GED-0507 is able to reduce body weight loss, ameliorate clinical and histological features of pulmonary fibrosis, and increase survival rate without any safety concerns. Here, we focused on the biomolecular effects of GED-0507 on various inflammatory/fibrotic pathways. We demonstrated that preventive and therapeutic administration of GED-0507 reduced the BLM-induced mRNA expression of several markers of fibrosis, including transforming growth factor (TGF)-ß, alpha-smooth muscle actin, collagen and fibronectin as well as epithelial-to-mesenchymal transition (EMT) and expression of mucin 5B. The beneficial effect of GED-0507 on pulmonary fibrosis was confirmed in vitro by its ability to control TGFß-induced myofibroblast activation in the A549 human alveolar epithelial cell line, the MRC-5 lung fibroblast line, and primary human lung fibroblasts. Compared with the US Food and Drug Administration-approved antifibrotic drugs pirfenidone and nintedanib, GED-0507 displayed greater antifibrotic activity by controlling alveolar epithelial cell dysfunction, EMT, and extracellular matrix remodeling. In conclusion, GED-0507 demonstrated potent antifibrotic properties and might be a promising drug candidate for the treatment of pulmonary fibrosis.

Botulinum toxin A promotes the transdifferentiation of primary keloid myofibroblasts into adipocyte-like cells.

Keloid is a type of unusually raised scar. Botulinum toxin A (BTX-A) has a great application potential in keloids treatment. Here, we investigated the functional role of BTX-A in keloids. We separated keloid tissues and normal skin tissues from keloid patients and found that the expression of myofibroblast markers, α-SMA, Collagen I, and Collagen III was increased in the keloid tissues as compared with normal skin tissues. Keloid fibroblasts derived from keloid tissues were treated with TGF-ß1 to induce the differentiation of fibroblasts into myofibroblasts. The keloid myofibroblasts displayed a significant up-regulation of α-SMA. BTX-A enhanced the expression of adipocyte markers, PPARγ and C/EBPα, and increased the accumulation of lipid droplets, and reduced the expression of α-SMA, Collagen I, and Collagen III in the keloid myofibroblasts. Moreover, BTX-A enhanced the expression of BMP4 and p-smad1/5/8. Noggin (BMP4 antagonist) treatment reversed BTX-A-mediated increase of PPARγ and C/EBPα expression and lipid droplets, and down-regulation of α-SMA, Collagen I, and Collagen III in primary keloid myofibroblasts. In conclusion, BTX-A promoted the transdifferentiation of primary keloid myofibroblasts into adipocyte-like cells, which may attribute to activate BMP4/Smad signalling pathway. Thus, this study provides new insights into the mechanism of BTX-A in keloid.

EHP-101 alleviates angiotensin II-induced fibrosis and inflammation in mice.

Some cannabinoids showed anti-inflammatory and antifibrotic activities. EHP-101 is an oral lipidic formulation of the novel non-psychotropic cannabidiol aminoquinone VCE-004.8, which showed antifibrotic activity in murine models of systemic sclerosis induced by bleomycin. We herein examined the effect of EHP-101 on cardiac and other organ fibrosis in a mouse model induced by Angiotensin II. VCE-004.8 inhibited TGFß- and Ang II-induced myofibroblast differentiation in cardiac fibroblasts detected by α-SMA expression. VCE-004.8 also inhibited Ang II-induced ERK 1 + 2 phosphorylation, NFAT activation and mRNA expression of IL1ß, IL6, Col1A2 and CCL2 in cardiac fibroblasts. Mice infused with Ang II resulted in collagen accumulation in left ventricle, aortic, dermal, renal and pulmonary tissues; oral administration of EHP-101, Ajulemic acid and Losartan improved these phenotypes. In myocardial tissue, Ang II induced infiltration of T cells and macrophages together with the accumulation of collagen and Tenascin C; those were all reduced by either EHP-101 or Losartan treatment. Cardiac tissue RNA-Seq analyses revealed a similar transcriptomic signature for both treatments for inflammatory and fibrotic pathways. However, the gene set enrichment analysis comparing data from EHP-101 vs Losartan showed specific hallmarks modified only by EHP-101. Specifically, EHP-101 inhibited the expression of genes such as CDK1, TOP2A and MKi67 that are regulated to the E2 factor family of transcription factors. This study suggests that the oral administration of EHP-101 prevents and inhibits cardiac inflammation and fibrosis. Furthermore, EHP-101 inhibits renal, pulmonary and dermal fibrosis. EHP-101 could offer new opportunities in the treatment of cardiac fibrosis and other fibrotic diseases.

CD147 mediates the CD44s-dependent differentiation of myofibroblasts driven by transforming growth factor-ß1.

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-ß1 and proinflammatory interleukin (IL)-1ß are widely associated with fibrotic progression. TGF-ß1 induces myofibroblast differentiation, while IL-1ß induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-ß1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-ß1- and IL-1ß-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-ß1-induced fibroblast/myofibroblast differentiation and IL-1ß-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell-cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.

Implant Fibrosis and the Underappreciated Role of Myofibroblasts in the Foreign Body Reaction.

Body implants and implantable medical devices have dramatically improved and prolonged the life of countless patients. However, our body repair mechanisms have evolved to isolate, reject, or destroy any object that is recognized as foreign to the organism and inevitably mounts a foreign body reaction (FBR). Depending on its severity and chronicity, the FBR can impair implant performance or create severe clinical complications that will require surgical removal and/or replacement of the faulty device. The number of review articles discussing the FBR seems to be proportional to the number of different implant materials and clinical applications and one wonders, what else is there to tell? We will here take the position of a fibrosis researcher (which, coincidentally, we are) to elaborate similarities and differences between the FBR, normal wound healing, and chronic healing conditions that result in the development of peri-implant fibrosis. After giving credit to macrophages in the inflammatory phase of the FBR, we will mainly focus on the activation of fibroblastic cells into matrix-producing and highly contractile myofibroblasts. While fibrosis has been discussed to be a consequence of the disturbed and chronic inflammatory milieu in the FBR, direct activation of myofibroblasts at the implant surface is less commonly considered. Thus, we will provide a perspective how physical properties of the implant surface control myofibroblast actions and accumulation of stiff scar tissue. Because formation of scar tissue at the surface and around implant materials is a major reason for device failure and extraction surgeries, providing implant surfaces with myofibroblast-suppressing features is a first step to enhance implant acceptance and functional lifetime. Alternative therapeutic targets are elements of the myofibroblast mechanotransduction and contractile machinery and we will end with a brief overview on such targets that are considered for the treatment of other organ fibroses.

Inhibition of CREB-CBP Signaling Improves Fibroblast Plasticity for Direct Cardiac Reprogramming.

Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) is a promising approach but remains a challenge in heart regeneration. Efforts have focused on improving the efficiency by understanding fundamental mechanisms. One major challenge is that the plasticity of cultured fibroblast varies batch to batch with unknown mechanisms. Here, we noticed a portion of in vitro cultured fibroblasts have been activated to differentiate into myofibroblasts, marked by the expression of αSMA, even in primary cell cultures. Both forskolin, which increases cAMP levels, and TGFß inhibitor SB431542 can efficiently suppress myofibroblast differentiation of cultured fibroblasts. However, SB431542 improved but forskolin blocked iCM reprogramming of fibroblasts that were infected with retroviruses of Gata4, Mef2c, and Tbx5 (GMT). Moreover, inhibitors of cAMP downstream signaling pathways, PKA or CREB-CBP, significantly improved the efficiency of reprogramming. Consistently, inhibition of p38/MAPK, another upstream regulator of CREB-CBP, also improved reprogramming efficiency. We then investigated if inhibition of these signaling pathways in primary cultured fibroblasts could improve their plasticity for reprogramming and found that preconditioning of cultured fibroblasts with CREB-CBP inhibitor significantly improved the cellular plasticity of fibroblasts to be reprogrammed, yielding ~2-fold more iCMs than untreated control cells. In conclusion, suppression of CREB-CBP signaling improves fibroblast plasticity for direct cardiac reprogramming.

TGFß promotes low IL10-producing ILC2 with profibrotic ability involved in skin fibrosis in systemic sclerosis.

OBJECTIVE: Innate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin fibrosis within systemic sclerosis (SSc). METHODS: Blood samples and skin biopsies from healthy donor or patients with SSc were analysed by immunostaining techniques. The fibrotic role of sorted ILC2 was studied in vitro on dermal fibroblast and further explored by transcriptomic approach. Finally, the efficacy of a new treatment against fibrosis was assessed with a mouse model of SSc. RESULTS: We found that ILC2 numbers were increased in the skin of patients with SSc and correlated with the extent of skin fibrosis. In SSc skin, KLRG1- ILC2 (natural ILC2) were dominating over KLRG1+ ILC2 (inflammatory ILC2). The cytokine transforming growth factor-ß (TGFß), whose activity is increased in SSc, favoured the expansion of KLRG1- ILC2 simultaneously decreasing their production of interleukin 10 (IL10), which regulates negatively collagen production by dermal fibroblasts. TGFß-stimulated ILC2 also increased myofibroblast differentiation. Thus, human KLRG1- ILC2 had an enhanced profibrotic activity. In a mouse model of SSc, therapeutic intervention-combining pirfenidone with the administration of IL10 was required to reduce the numbers of skin infiltrating ILC2, enhancing their expression of KLRG1 and strongly alleviating skin fibrosis. CONCLUSION: Our results demonstrate a novel role for natural ILC2 and highlight their inter-relationships with TGFß and IL10 in the development of skin fibrosis, thereby opening up new therapeutic approaches in SSc.