Fibroblast Extracellular Matrix Research Articles

Inhibition of STAT3 by S3I-201 suppress peritoneal fibroblast phenotype conversion and alleviate peritoneal fibrosis.

Peritoneal fibrosis is a common pathological response to long-term peritoneal dialysis (PD) and a major cause for PD discontinuation. Understanding the cellular and molecular mechanisms underlying the induction and progression of peritoneal fibrosis is of great interest. In our study, invitro study revealed that signal transducer and activator of transcription 3 (STAT3) is a key factor in fibroblast activation and extracellular matrix (ECM) synthesis. Furthermore, STAT3 induced by IL-6 trans-signalling pathway mediate the fibroblasts of the peritoneal stroma contributed to peritoneal fibrosis. Inhibition of STAT3 exerts an antifibrotic effect by attenuating fibroblast activation and ECM production with an invitro co-culture model. Moreover, STAT3 plays an important role in the peritoneal fibrosis in an animal model of peritoneal fibrosis developed in mice. Blocking STAT3 can reduce the peritoneal morphological changes induced by chlorhexidine gluconate. In conclusion, our findings suggested STAT3 signalling played an important role in peritoneal fibrosis. Therefore, blocking STAT3 might become a potential treatment strategy in peritoneal fibrosis.

Abstract 285: Targeting TXNDC5 in stromal fibroblasts resolves desmoplasia and resistance to immune checkpoint blockade in mesenchymal-type colorectal cancer

Abstract Objectives: Mesenchymal-type colorectal cancer (CRC), characterized by strong stromal infiltration and immune tolerance, resists immune checkpoint blockade and has poor outcomes. Cancer-associated fibroblasts (CAFs), abundant in tumor stroma, actively remodel the extracellular matrix (ECM), modulate immune evasion, and drive tumor progression. We have recently identified thioredoxin domain-containing protein 5 (TXNDC5), a protein disulfide isomerase (PDI), as a critical mediator of fibroblast activation and ECM remodeling in organ fibrosis. We hypothesized that TXNDC5 could contribute to fibroblast activation, stroma formation and disease progression in cancer, especially in the stroma-enriched fibrogenic mesenchymal-type CRC. Methods: Transcriptome databases of CRC were re-analyzed to determine the clinical relevance of TXNDC5. Experimentally, CRC was induced in mouse lines by azoxymethane (AOM) and dextran sulfate sodium (DSS) stimuli, a model sharing multiple characteristics with human mesenchymal-type CRC. Human colonic fibroblast line CCD-18co was used to investigate the molecular mechanisms by which TXNDC5 regulates colonic fibroblast activities. Fibroblast-specific TXNDC5 knockout (Col1a2-Cre/ERT2*TXNDC5fl/fl) mice were generated, combining with single-cell RNA sequencing analyses on AOM/DSS-induced CRC tumors in these animals, to clarify how fibroblast TXNDC5 impact tumor microenvironment, CRC progression and response to immune checkpoint blockade. Findings: TXNDC5 was predominantly expressed in stromal fibroblasts of human and mouse CRC. Fibroblast-specific deletion of Txndc5 lessened CAF activation, attenuated tumor fibrosis and reduced tumor burden in AOM/DSS-induced CRC. Mechanistically, increased TXNDC5 levels augments TGFβ signaling in CAF by post-translational stabilization of TGFBR1 through its PDI activity. In addition, deletion of Txndc5 in CAFs led to less tumor desmoplasia, decompressed tumor vessels and attenuated intra-tumoral hypoxia, thereby easing immune tolerance and increasing cytotoxic T cell infiltration in CRC. Single-cell transcriptome analysis revealed a marked change of intra-tumoral immune cell populations upon fibroblast-specific deletion of TXNDC5, shifting from myeloid-derived suppressive cells to cytotoxic tumor-infiltrating lymphocytes. Importantly, depletion of TXNDC5 in CAFs potentiated the anti-tumor effects of immune checkpoint blockade with anti-PD1 therapy in CRC. Conclusions: Our data suggest an important yet previously unrecognized role of fibroblast TXNDC5 in CRC progression, through enhancing CAF activation, stroma formation and immune escape. Combining immune checkpoint blockade with TXNDC5 deletion synergistically improved anti-tumor effects in CRC. Targeting TXNDC5, therefore, can be a novel therapeutic approach for CRC patients. Citation Format: Kai-Lin Cheng, Chih-I Chen, Shu-Han Yu, Huai-Wen Liang, Yi-Wei Tsai, Chen-Ting Hung, Yu-Shan Lin, Yi-Shiuan Tzeng, Sung-Jan Lin, Yueh-Feng Wu, Jen-Kuang Lee, Chia-Hui Yu, Shuei-Liong Lin, Shih-Yu Chen, Tzu-Tang Wei, Yun-Ju Huang, Ruey-Hwa Chen, Ching-Chow Chen, Kai-Chien Yang. Targeting TXNDC5 in stromal fibroblasts resolves desmoplasia and resistance to immune checkpoint blockade in mesenchymal-type colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 285.

Characterization of cardiac fibroblast-extracellular matrix crosstalk across developmental ages provides insight into age-related changes in cardiac repair.

Heart failure afflicts an estimated 6.5 million people in the United States, driven largely by incidents of coronary heart disease (CHD). CHD leads to heart failure due to the inability of adult myocardial tissue to regenerate after myocardial infarction (MI). Instead, immune cells and resident cardiac fibroblasts (CFs), the cells responsible for the maintenance of the cardiac extracellular matrix (cECM), drive an inflammatory wound healing response, which leads to fibrotic scar tissue. However, fibrosis is reduced in fetal and early (<1-week-old) neonatal mammals, which exhibit a transient capability for regenerative tissue remodeling. Recent work by our laboratory and others suggests this is in part due to compositional differences in the cECM and functional differences in CFs with respect to developmental age. Specifically, fetal cECM and CFs appear to mitigate functional loss in MI models and engineered cardiac tissues, compared to adult CFs and cECM. We conducted 2D studies of CFs on solubilized fetal and adult cECM to investigate whether these age-specific functional differences are synergistic with respect to their impact on CF phenotype and, therefore, cardiac wound healing. We found that the CF migration rate and stiffness vary with respect to cell and cECM developmental age and that CF transition to a fibrotic phenotype can be partially attenuated in the fetal cECM. However, this effect was not observed when cells were treated with cytokine TGF-β1, suggesting that inflammatory signaling factors are the dominant driver of the fibroblast phenotype. This information may be valuable for targeted therapies aimed at modifying the CF wound healing response and is broadly applicable to age-related studies of cardiac remodeling.

GOLM1 Promotes Pulmonary Fibrosis through Upregulation of NEAT1.

Idiopathic pulmonary fibrosis (IPF) is a lethal progressive disease with elusive molecular mechanisms and limited therapeutic options. Aberrant activation of fibroblasts is a central hallmark of lung fibrosis. Here, we report that Golgi membrane protein 1 (GOLM1, also known as GP73 or GOLPH2) was increased in the lungs of patients with pulmonary fibrosis and mice with bleomycin (BLM)-induced pulmonary fibrosis. Loss of GOLM1 inhibited proliferation, differentiation, and extracellular matrix deposition of fibroblasts, whereas overexpression of GOLM1 exerted the opposite effects. Similarly, worsening pulmonary fibrosis after BLM treatment was observed in GOLM1-knock-in mice, whereas BLM-treated Golm1-knockout mice exhibited alleviated pulmonary fibrosis and collagen deposition. Furthermore, we identified long noncoding RNA NEAT1 downstream of GOLM1 as a potential mediator of pulmonary fibrosis through increased GOLM1 expression. Depletion of NEAT1 inhibited fibroblast proliferation and extracellular matrix production and reversed the profibrotic effects of GOLM1 overexpression. Additionally, we identified KLF4 as a downstream mediator of GOLM1 signaling to NEAT1. Our findings suggest that GOLM1 plays a pivotal role in promoting pulmonary fibrosis through the GOLM1-KLF4-NEAT1 signaling axis. Targeting GOLM1 and its downstream pathways may represent a novel therapeutic strategy for treating pulmonary fibrosis.

The upregulation of circFoxp1 influences keloid by promoting cell proliferation.

As a result of abnormal wound healing in susceptible individuals, keloids are characterized by hyperproliferation of fibroblasts and excessive deposition of the extracellular matrix (ECM). Current surgical and therapeutic modalities provide limited satisfactory results. Circular ribonucleic acids (circRNAs) play a crucial role in the pathogenesis of various fibrotic diseases, but the potential biological function and expression profile of circRNAs in keloid formation remain unknown. In this study, we explored the function of circFoxp1 on keloid formation. Methods: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results revealed that circFoxp1 expression was higher in the keloid tissues. Furthermore, RNA-fluorescence in situ hybridization (RNA-FISH) and RNAscope illustrated that circFoxp1 was present in the cytoplasm. Subsequent cellular experiments demonstrated that circFoxp1 overexpression enhanced proliferation, migration, and ECM deposition. In addition, apoptosis was inhibited. Cell proliferation, inflammatory response, and oxidative phosphorylation of fibroblasts were also observed by RNA sequencing and were closely related to scar formation. The therapeutic potential of circFoxp1 was investigated by establishing keloid implantation models. In vivo, circFoxp1 can promote fibroblast proliferation and ECM deposition. RNA pull-down and western blot assays verified the interaction of circFoxp1 with RACK1. The present study reveals that circFoxp1 contributes to the pathological hyperplasia of keloid, which may improve inflammation and cell proliferation. Our data indicate that circFoxp1 may serve as a novel, promising therapeutic target, presenting a new avenue for understanding the underlying pathogenesis of keloid.

Biomimetic Cardiac Fibrotic Model for Antifibrotic Drug Screening.

Cardiac fibrosis is characterized by pathological proliferation and activation of cardiac fibroblasts to myofibroblasts. Inhibition and reverse of transdifferentiation of cardiac fibroblasts to myofibroblasts is a potential strategy for cardiac fibrosis. Despite substantial progress, more effort is needed to discover effective drugs to improve and reverse cardiac fibrosis. The main reason for the slow development of antifibrotic drugs is that the traditional polystyrene culture platform does not recapitulate the microenvironment where cells reside in tissues. In this study, we propose an in vitro cardiac fibrotic model by seeding electrospun yarn scaffolds with cardiac fibroblasts. Our results show that yarn scaffolds allow three-dimensional growth of cardiac fibroblasts, promote extracellular matrix (ECM) deposition, and induce the transdifferentiation of cardiac fibroblasts to myofibroblasts. Exogenous transforming growth factor-β1 further promotes cardiac fibroblast activation and ECM deposition, which makes it a suitable fibrotic model to predict the antifibrotic potential of drugs. By using this platform, we demonstrate that both Honokiol (HKL) and Pirfenidone (PFD) show potential in antifibrosis to some extent. HKL is more efficient in antifibrosis than PFD as revealed by biochemical composition, gene, and molecular analyses as well as histological and biomechanical analysis. The electrospun yarn scaffold provides a novel platform for constructing in vitro fibrotic models to study cardiac fibrosis and to predict the antifibrotic efficacy of novel drugs.

Role of the epithelial barrier in intestinal fibrosis associated with inflammatory bowel disease: relevance of the epithelial-to mesenchymal transition.

In inflammatory bowel disease (IBD), chronic inflammation in the gastrointestinal tract can lead to tissue damage and remodelling, which can ultimately result in fibrosis. Prolonged injury and inflammation can trigger the activation of fibroblasts and extracellular matrix (ECM) components. As fibrosis progresses, the tissue becomes increasingly stiff and less functional, which can lead to complications such as intestinal strictures, obstructive symptoms, and eventually, organ dysfunction. Epithelial cells play a key role in fibrosis, as they secrete cytokines and growth factors that promote fibroblast activation and ECM deposition. Additionally, epithelial cells can undergo a process called epithelial-mesenchymal transition, in which they acquire a more mesenchymal-like phenotype and contribute directly to fibroblast activation and ECM deposition. Overall, the interactions between epithelial cells, immune cells, and fibroblasts play a critical role in the development and progression of fibrosis in IBD. Understanding these complex interactions may provide new targets for therapeutic interventions to prevent or treat fibrosis in IBD. In this review, we have collected and discussed the recent literature highlighting the contribution of epithelial cells to the pathogenesis of the fibrotic complications of IBD, including evidence of EMT, the epigenetic control of the EMT, the potential influence of the intestinal microbiome in EMT, and the possible therapeutic strategies to target EMT. Finally we discuss the pro-fibrotic interactions epithelial-immune cells and epithelial-fibroblasts cells.

M6A-modified miR-143-3p inhibits epithelial mesenchymal transition in bronchial epithelial cells and extracellular matrix production in lung fibroblasts by targeting Smad3

BackgroundAirway epithelial cells epithelial mesenchymal transition (EMT) and lung fibroblasts extracellular matrix (ECM) production are the key steps in airway remodeling. Our previous study demonstrated that miR-143-3p has the ability to impede airway smooth muscle cell proliferation and ECM deposition. However, the function of miR-143-3p in airway epithelial cells and lung fibroblasts remains unclear. MethodsCell viability was determined using MTT method, while cell migration was evaluated through scratch assay. EMT and ECM proteins were detected by western blot, RT-qPCR, and ELISA. To determine the level of miR-143-3p m6A methylation, we employed the meRIP-qPCR assay. Additionally, the binding of miR-143-3p with Smad3 were projected by bioinformatics and validated by dual luciferase reporter assays. ResultsIt was discovered that the expression of miR-143-3p were lower in both asthma patients and TGF-β1-treated human bronchial epithelial 16HBE cells and human lung fibroblast HPF cells. Upregulation of miR-143-3p restrained 16HBE cell migration, and decreased EMT mesenchymal markers and increased epithelial markers. And upregulation of miR-143-3p impaired cell viability and ECM protein production in HPF cells. Mechanistically, interfering with METTL3 resulted in decreased m6A modification of miR-143-3p and led to lower levels of miR-143-3p. Moreover, miR-143-3p were verified to directly target and downregulate Smad3. Upregulation of Smad3 attenuated the effects of miR-143-3p on cell EMT and ECM production. ConclusionMiR-143-3p inhibits airway epithelial cell EMT as well as lung fibroblast ECM production by downregulating Smad3. Therefore, miR-143-3p may be a promising target to reduce airway remodeling in asthma.

Unlocking the post-transplant microenvironment for successful islet function and survival.

Islet transplantation (IT) offers the potential to restore euglycemia for patients with type 1 diabetes mellitus (T1DM). Despite improvements in islet isolation techniques and immunosuppressive regimes, outcomes remain suboptimal with UK five-year graft survivals (5YGS) of 55% and most patients still requiring exogenous insulin after multiple islet infusions. Native islets have a significant non-endocrine component with dense extra-cellular matrix (ECM), important for islet development, cell survival and function. Collagenase isolation necessarily disrupts this complex islet microenvironment, leaving islets devoid of a supporting framework and increasing vulnerability of transplanted islets. Following portal venous transplantation, a liver injury response is potentially induced, which typically results in inflammation and ECM deposition from liver specific myofibroblasts. The impact of this response may have important impact on islet survival and function. A fibroblast response and ECM deposition at the kidney capsule and eye chamber alongside other implantation sites have been shown to be beneficial for survival and function. Investigating the implantation site microenvironment and the interactions of transplanted islets with ECM proteins may reveal therapeutic interventions to improve IT and stem-cell derived beta-cell therapy.

Profibrotic role of the SOX9–MMP10–ECM biosynthesis axis in the tracheal fibrosis after injury and repair

Fibroblast activation and extracellular matrix (ECM) deposition play an important role in the tracheal abnormal repair process and fibrosis. As a transcription factor, SOX9 is involved in fibroblast activation and ECM deposition. However, the mechanism of how SOX9 regulates fibrosis after tracheal injury remains unclear. We investigated the role of SOX9 in TGF-β1-induced fibroblast activation and ECM deposition in rat tracheal fibroblast (RTF) cells. SOX9 overexpression adenovirus (Ad-SOX9) and siRNA were transfected into RTF cells. We found that SOX9 expression was up-regulated in RTF cells treated with TGF-β1. SOX9 overexpression activated fibroblasts and promoted ECM deposition. Silencing SOX9 inhibited cell proliferation, migration, and ECM deposition, induced G2 arrest, and increased apoptosis in RTF cells. RNA-seq and chromatin immunoprecipitation sequencing (ChIP-seq) assays identified MMP10, a matrix metalloproteinase involved in ECM deposition, as a direct target of SOX9, which promotes ECM degradation by increasing MMP10 expression through the Wnt/β-catenin signaling pathway. Furthermore, in vivo, SOX9 knockdown ameliorated granulation proliferation and tracheal fibrosis, as manifested by reduced tracheal stenosis. In conclusion, our findings indicate that SOX9 can drive fibroblast activation, cell proliferation, and apoptosis resistance in tracheal fibrosis via the Wnt/β-catenin signaling pathway. The SOX9–MMP10–ECM biosynthesis axis plays an important role in tracheal injury and repair. Targeting SOX9 and its downstream target MMP10 may represent a promising therapeutic approach for tracheal fibrosis.

Matrix stiffness-induced α-tubulin acetylation is required for skin fibrosis formation through activation of Yes-associated protein.

Skin fibrosis, a pathological process featured by fibroblast activation and extracellular matrix (ECM) deposition, makes a significant contribution to morbidity. Studies have identified biomechanics as the central element in the complex network of fibrogenesis that drives the profibrotic feedback loop. In this study, we found that the acetylation of α-tubulin at lysine 40 (K40) was augmented in fibrotic skin tissues. Further analysis showed that α-tubulin acetylation is required for fibroblast activation, including contraction, migration, and ECM deposition. More importantly, we revealed that biomechanics-induced upregulation of K40 acetylation promotes fibrosis by mediating mechanosensitive Yes-associated protein S127 dephosphorylation and its cytoplasm nucleus shuttle. Furthermore, we demonstrated that the knockdown of α-tubulin acetyltransferase 1 could rescue the K40 acetylation upregulation caused by increased matrix rigidity and ameliorate skin fibrosis both in vivo and in vitro. Herein, we highlight the critical role of α-tubulin acetylation in matrix stiffness-induced skin fibrosis and clarify a possible molecular mechanism. Our research suggests α-tubulin acetylation as a potential target for drug design and therapeutic intervention.

P53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload.

Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure. We used the Tcf21 (transcription factor 21)MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction. Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21-lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation. This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.

Advancements in adipose-derived stem cell therapy for skin fibrosis

Pathological scarring and scleroderma, which are the most common conditions of skin fibrosis, pathologically manifest as fibroblast proliferation and extracellular matrix (ECM) hyperplasia. Fibroblast proliferation and ECM hyperplasia lead to fibrotic tissue remodeling, causing an exaggerated and prolonged wound-healing response. The pathogenesis of these diseases has not been fully clarified and is unfortunately accompanied by exceptionally high medical needs and poor treatment effects. Currently, a promising and relatively low-cost treatment has emerged-adipose-derived stem cell (ASC) therapy as a branch of stem cell therapy, including ASCs and their derivatives-purified ASC, stromal vascular fraction, ASC-conditioned medium, ASC exosomes, etc., which are rich in sources and easy to obtain. ASCs have been widely used in therapeutic settings for patients, primarily for the defection of soft tissues, such as breast enhancement and facial contouring. In the field of skin regeneration, ASC therapy has become a hot research topic because it is beneficial for reversing skin fibrosis. The ability of ASCs to control profibrotic factors as well as anti-inflammatory and immunomodulatory actions will be discussed in this review, as well as their new applications in the treatment of skin fibrosis. Although the long-term effect of ASC therapy is still unclear, ASCs have emerged as one of the most promising systemic antifibrotic therapies under development.

Pancreatic cancer-derived small extracellular vesical ezrin activates fibroblasts to exacerbate cancer metastasis through STAT3 and YAP-1 signaling pathways.

Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC), play an important role in tumorigenesis, metastasis, and chemoresistance. Tumor-derived small extracellular vesicles (sEVs), which mediate cell-to-cell communication between cancer cells and fibroblasts, are also critical for cancer progression and metastasis. However, it remains unclear how PDAC cell-derived sEVs activate fibroblasts, which contributes to tumor progression. Here, we report that ezrin (EZR) expression in PDAC cell-derived sEVs (sEV-EZR) can activate fibroblasts, resulting in increased migration ability and high expression of α-SMA, PDGFRB, and high production of extracellular matrix in fibroblasts. Reciprocally, sEV-EZR-activated fibroblasts enhanced PDAC cell proliferation, invasion, and metastasis to the liver in animal models. Conversely, fibroblasts treated with PDAC cell-derived sEVs with EZR knockdown resulted in the reduced metastatic ability of PDAC. Mechanistically, we demonstrated that PDAC cell-derived sEV-EZR increases the STAT3 and YAP-1 signaling pathways to induce fibroblast activation, and the activated fibroblasts promote PDAC cell proliferation, invasion, and liver metastasis. Inhibition of the STAT3 and YAP-1 signaling pathways by gene knockdown can abrogate sEV-EZR-induced effects. These findings suggest that targeting the interaction between PDAC cell-derived sEV-EZR and fibroblasts is a potential therapeutic strategy for PDAC.

Exploring the Potential of Ultrasound Therapy to Reduce Skin Scars: An In Vitro Study Using a Multi-Well Device Based on Printable Piezoelectric Transducers.

Excessive skin scarring affects over 100 million patients worldwide, with effects ranging from cosmetic to systemic problems, and an effective treatment is yet to be found. Ultrasound-based therapies have been used to treat a variety of skin disorders, but the exact mechanisms behind the observed effects are still unclear. The aim of this work was to demonstrate the potential of ultrasound for the treatment of abnormal scarring by developing a multi-well device based on printable piezoelectric material (PiezoPaint™). First, compatibility with cell cultures was evaluated using measurements of heat shock response and cell viability. Second, the multi-well device was used to treat human fibroblasts with ultrasound and quantify their proliferation, focal adhesions, and extracellular matrix (ECM) production. Ultrasound caused a significant reduction in fibroblast growth and ECM deposition without changes in cell viability or adhesion. The data suggest that these effects were mediated by nonthermal mechanisms. Interestingly, the overall results suggest that ultrasound treatment would a be beneficial therapy for scar reduction. In addition, it is expected that this device will be a useful tool for mapping the effects of ultrasound treatment on cultured cells.

Loss of RhoE Function in Dermatofibroma Promotes Disorganized Dermal Fibroblast Extracellular Matrix and Increased Integrin Activation

Dermatofibromas are common, benign fibrous skin tumors that can occur at any skin site. In most cases, dermatofibromas are solitary and sporadic, but a few are multiple and familial, and the mechanisms leading to these lesions are currently unclear. Using exome sequencing, we have identified a heterozygous variant in a pedigree with autosomal dominant multiple familial dermatofibromas within RND3 (c.692C>T,p.T231M) that encodes for the small GTPase RhoE, a regulator of the actin cytoskeleton. Expression of T231M-RhoE or RhoE depletion using CRISPR in human dermal fibroblasts increased proliferation, adhesion to extracellular matrix via enhanced β1 integrin activation and more disorganized matrix. The enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was identified as a binding partner for RhoE and formation of this complex was disrupted by T231M-RhoE. PLOD2 promotes collagen crosslinking and activation of β1 integrins, and depleting PLOD2 in T231M-RhoE expressing cells reduced T231M-RhoE-mediated β1 integrin activation and led to increased matrix alignment. Immunohistochemical analysis revealed reduced expression of RhoE but increased expression of PLOD2 in the dermis of dermatofibroma skin samples compared to controls. Our data show loss of RhoE function leads to increased PLOD2 activation, enhancing integrin activation and leading to disorganized extracellular matrix, contributing to dermatofibroma.

GATA6 triggers fibroblast activation and tracheal fibrosis through the Wnt/β-catenin pathway

Tracheal fibrosis is a key abnormal repair process leading to fatal stenosis, characterized by excessive fibroblast activation and extracellular matrix (ECM) deposition. GATA6, a zinc finger-containing transcription factor, is involved in fibroblast activation, while its role in tracheal fibrosis remains obscure. The present study investigated the potential role of GATA6 as a novel regulator of tracheal fibrosis. It was found that GATA6 and α-smooth muscle actin (α-SMA) were obviously increased in tracheal fibrotic granulations and in TGFβ1-treated primary tracheal fibroblasts. GATA6 silencing inhibited TGFβ1-stimulated fibroblast proliferation and ECM synthesis, promoted cell apoptosis, and inactivated Wnt/β-catenin pathway, whereas GATA6 overexpression showed the reverse effects. SKL2001, an agonist of Wnt/β-catenin signaling, restored collagen1a1 and α-SMA expression which was suppressed by GATA6 silencing. Furthermore, in vivo, knockdown of GATA6 ameliorated tracheal fibrosis, as manifested by reduced tracheal stenosis and ECM deposition. GATA6 inhibition in rat tracheas also impaired granulation proliferation, increased apoptosis, and inactivated Wnt/β-catenin pathway. In conclusion, our findings indicate that GATA6 triggers fibroblast activation, cell proliferation, and apoptosis resistance in tracheal fibrosis via the Wnt/β-catenin signaling pathway. Targeting GATA6 may represent a promising therapeutic approach for tracheal fibrosis.

A novel molecular mechanism of vascular fibrosis in Takayasu arteritis: macrophage-derived GPNMB promoting adventitial fibroblast extracellular matrix production in the aorta

Takayasu arteritis (TAK) is a chronic large vessel disease characterized by aortic fibrotic thickening, which was mainly mediated by activation of aorta adventitial fibroblasts (AAFs). Our previous genetic study demonstrated that TAK-associated locus IL6 rs2069837 regulated glycoprotein non-metastatic melanoma protein B (GPNMB) expression. Thus, this study aimed to investigate the pathogenic role of GPNMB in TAK. Through pathological staining, we find that GPNMB was mainly expressed in vascular adventitia and positively correlated with adventitial extracellular matrix (ECM) expression in TAK vascular lesion. Specifically, GPNMB was increased in adventitial CD68+ macrophages, which were closely located with CD90+ adventitial fibroblasts. In in-vitro cell culture, THP-1-derived macrophages with GPNMB overexpression promoted ECM expression in AAFs. This effect was also confirmed in aortic tissue or AAFs culture with GPNMB overexpression or active GPNMB protein stimulation. Mechanistically, Co-IP assay and siRNA or inhibitor intervention demonstrated that integrin αVβ1 receptor mediated GPNMB effect on AAFs, which also activated downstream Akt and Erk pathway in AAFs. Furthermore, we showed that leflunomide treatment inhibited GPNMB-mediated fibrosis in AAFs, as well as GPNMB expression in macrophages, which were also partially validated in leflunomide-treated patients. Taken together, these data indicated that macrophage-derived GPNMB promotes AAFs ECM expression via the integrin αVβ1 receptor and Akt/Erk signaling pathway and leflunomide might play an anti-fibrotic role in TAK by interfering with the macrophage-derived GPNMB/AAFs axis. This study provides evidence that targeting GPNMB is a potential therapeutic strategy for treating vascular fibrosis in TAK.

Abstract 15133: Matricellular Protein CCN1 Regulates Cardiac Fibroblast ECM Synthesis and Alignment

Introduction: Cellular communication network (CCN) proteins constitute a 6-member family of extracellular matrix (ECM)-associated signaling proteins that have important roles in wound healing and tissue fibrosis. Though their function in the heart is not fully explicated, CCN1 is often indicated as an antifibrotic factor. Yet, CCN1 levels correlate with infarct size and ventricular dysfunction in myocardial infarct patients—signifying a pro-fibrotic role for this factor in the heart. Objective: To determine the contribution of endogenous CCN1 to fibroblast (FB) ECM synthesis and organization. Methods: In vitro murine cardiac FB ECM synthesis, activation, proliferation, and matrix organization was evaluated in cells treated with CCN1 or non-target (NT) siRNA, by qPCR/immunoblotting for collagens and synthetic enzymes, EdU incorporation, and type I collagen immunostaining of decellularized matrices, respectively. CCN1-dependent integrin pathways modulating FB collagen synthetic enzyme expression were identified and interrogated via in vitro immunoprecipitation and RNAi-mediated depletion. Results: CCN1-deficient FBs showed reduced expression of type I collagen and collagen processing enzymes ( P4ha1 , P4hb , Plod1 , Plod2 , Loxl2 ). siRNA-mediated knockdown of CCN1 did not impact FB activation (α-SMA), but significantly reduced the expression of collagen synthetic/processing enzymes ( P4ha1 , Plod1 , Plod2 , Loxl2 ). Deficiency of CCN1 in both unstimulated and TGFβ1-stimulated FBs resulted in reduced proliferative capacity (7.17%, p=0.0205 and 5.34%, p=0.0428, respectively, relative to NT, n=3). CCN1 depletion promoted the alignment of FB-derived collagen fibers (0.30 ± 0.03 vs. 0.58 ± 0.04; mean ± SEM, p&lt;0.0001, n=6). An interaction between CCN1 and integrin αvβ3 was validated; moreover, knockdown of integrin β3 reduced the expression of enzymes associated with collagen cross-linking ( Plod1 , Plod2 , Loxl2 ), partly phenocopying CCN1 depletion. Conclusions: The data suggest that CCN1 drives pro-fibrotic signaling and limits ECM organization in cardiac FBs. This may have implications for CCN1 in the infarcted heart by promoting scar establishment; however, long-term expression could lead to progressive detrimental remodeling.

Absorbable Electrospun Poly-4-hydroxybutyrate Scaffolds as a Potential Solution for Pelvic Organ Prolapse Surgery.

Women with pelvic organ prolapse (POP) have bothersome complaints that significantly affect their quality of life. While native tissue repair is associated with high recurrence rates, polypropylene knitted implants have caused specific implant-related adverse events that have detrimental, often irreversible, effects. We hypothesize that surgical outcome can be improved with a tissue-engineered solution using an absorbable implant that mimics the natural extracellular matrix (ECM) structure, releases estrogen, and activates collagen metabolism by fibroblasts as the main regulators of wound healing. To this aim, we produced electrospun poly-4-hydroxybutyrate (P4HB) scaffolds and biofunctionalized them with estradiol (E2). The cell-implant interactions relevant for POP repair were assessed by seeding primary POP vaginal fibroblasts isolated from patients on electrospun P4HB scaffolds with 1%, 2%, or 5% E2 and without E2. To test our hypothesis on whether ECM mimicking structures should improve regeneration, electrospun P4HB was compared to knitted P4HB implants. We evaluated vaginal fibroblast proliferation, ECM deposition, and metabolism by quantification of collagen, elastin, and matrix metalloproteinases and by gene expression analysis for 28 days. We established effective E2 drug loading with a steady release over time. Significantly higher cell proliferation, collagen-, and elastin deposition were observed on electrospun P4HB scaffolds as compared to knitted P4HB. For this study, physical properties of the scaffolds were more determinant on the cell response than the release of E2. These results indicate that making these electrospun P4HB scaffolds E2-releasing appears to be technically feasible. In addition, electrospun P4HB scaffolds promote the cellular response of vaginal fibroblasts and further studies are merited to assess if their use results in improved surgical outcomes in case of POP repair.