Excessive Extracellular Matrix Research Articles

Inhibitory Effects of 3-Cyclopropylmethoxy-4-(difluoromethoxy) Benzoic Acid on TGF-β1-Induced Epithelial-Mesenchymal Transformation of In Vitro and Bleomycin-Induced Pulmonary Fibrosis In Vivo.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease characterized by lung inflammation and excessive deposition of extracellular matrix components. Transforming growth factor-β1 (TGF-β1) induced epithelial-mesenchymal transformation of type 2 lung epithelial cells leads to excessive extracellular matrix deposition, which plays an important role in fibrosis. Our objective was to evaluate the effects of 3-cyclopropylmethoxy-4-(difluoromethoxy) benzoic acid (DGM) on pulmonary fibrosis and aimed to determine whether EMT plays a key role in the pathogenesis of pulmonary fibrosis and whether EMT can be used as a therapeutic target for DGM therapy to reduce IPF. Firstly, stimulation of in vitro cultured A549 cells to construct EMTs with TGF-β1. DGM treatment inhibited the expression of proteins such as α-SMA, vimentin, and collagen Ⅰ and increased the expression of E-cadherin. Accordingly, Smad2/3 phosphorylation levels were significantly reduced by DGM treatment. Secondly, models of tracheal instillation of bleomycin and DGM were used to treat rats to demonstrate their therapeutic effects, such as improving lung function, reducing lung inflammation and fibrosis, reducing collagen deposition, and reducing the expression of E-cadherin. In conclusion, DGM attenuates TGF-β1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in rats.

High-Fat Diet Related Lung Fibrosis-Epigenetic Regulation Matters

Pulmonary fibrosis (PF) is an interstitial lung disease characterized by the destruction of the pulmonary parenchyma caused by excessive extracellular matrix deposition. Despite the well-known etiological factors such as senescence, aberrant epithelial cell and fibroblast activation, and chronic inflammation, PF has recently been recognized as a metabolic disease and abnormal lipid signature was observed both in serum and bronchoalveolar lavage fluid (BALF) of PF patients and mice PF model. Clinically, observational studies suggest a significant link between high-fat diet (HFD) and PF as manifested by high intake of saturated fatty acids (SFAs) and meat increases the risk of PF and mice lung fibrosis. However, the possible mechanisms between HFD and PF remain unclear. In the current review we emphasize the diversity effects of the epigenetic dysregulation induced by HFD on the fibrotic factors such as epithelial cell injury, abnormal fibroblast activation and chronic inflammation. Finally, we discuss the potential ways for patients to improve their conditions and emphasize the prospect of targeted therapy based on epigenetic regulation for scientific researchers or drug developers.

Regulation of Mesenchymal Cell Fate by Transfer of Active Gasdermin-D via Monocyte-Derived Extracellular Vesicles.

Fibrosis is characterized by inappropriately persistent myofibroblast accumulation and excessive extracellular matrix deposition with the disruption of tissue architecture and organ dysfunction. Regulated death of reparative mesenchymal cells is critical for normal wound repair, but profibrotic signaling promotes myofibroblast resistance to apoptotic stimuli. A complex interplay between immune cells and structural cells underlies lung fibrogenesis. However, there is a paucity of knowledge on how these cell populations interact to orchestrate physiologic and pathologic repair of the injured lung. In this context, gasdermin-D (GsdmD) is a cytoplasmic protein that is activated following cleavage by inflammatory caspases and induces regulated cell death by forming pores in cell membranes. This study was undertaken to evaluate the impact of human (Thp-1) monocyte-derived extracellular vesicles and GsdmD on human lung fibroblast death. Our data show that active GsdmD delivered by monocyte-derived extracellular vesicles induces caspase-independent fibroblast and myofibroblast death. This cell death was partly mediated by GsdmD-independent induction of cellular inhibitor of apoptosis 2 (cIAP-2) in the recipient fibroblast population. Our findings, to our knowledge, define a novel paradigm by which inflammatory monocytes may orchestrate the death of mesenchymal cells in physiologic wound healing, illustrating the potential to leverage this mechanism to eliminate mesenchymal cells and facilitate the resolution of fibrotic repair.

BRD4 promotes hepatic stellate cells activation and hepatic fibrosis via mediating P300/H3K27ac/PLK1 axis

Hepatic fibrosis (HF) is a reversible wound-healing response characterized by excessive extracellular matrix (ECM) deposition and secondary to persistent chronic injury. Bromodomain protein 4 (BRD4) commonly functions as a “reader” to regulate epigenetic modifications involved in various biological and pathological events, but the mechanism of HF remains unclear. In this study, we established a CCl4-induced HF model and spontaneous recovery model in mice and found aberrant BRD4 expression, which was consistent with the results in human hepatic stellate cells (HSCs)- LX2 cells in vitro. Subsequently, we found that distriction and inhibition of BRD4 restrained TGFβ-induced trans-differentiation of LX2 cells into activated, proliferative myofibroblasts and accelerated apoptosis, and BRD4 overexpression blocked MDI-induced LX2 cells inactivation and promoted the proliferation and inhibited apoptosis of inactivated cells. Additionally, adeno-associated virus serotype 8-loaded short hairpin RNA-mediated BRD4 knockdown in mice significantly attenuated CCl4-induced fibrotic responses including HSCs activation and collagen deposition. Mechanistically, BRD4 deficiency inhibited PLK1 expression in activated LX2 cells, and ChIP and Co-IP assays revealed that BRD4 regulation of PLK1 was dependent on P300-mediated acetylation modification for H3K27 on the PLK1 promoter. In conclusion, BRD4 deficiency in the liver alleviates CCl4-induced HF in mice, and BRD4 participates in the activation and reversal of HSCs through positively regulating the P300/H3K27ac/PLK1 axis, providing a potential insight for HF therapy.

Activation of the Hedgehog signaling pathway leads to fibrosis in aortic valves

BackgroundFibrosis is a pathological wound healing process characterized by excessive extracellular matrix deposition, which interferes with normal organ function and contributes to ~ 45% of human mortality. Fibrosis develops in response to chronic injury in nearly all organs, but the a cascade of events leading to fibrosis remains unclear. While hedgehog (Hh) signaling activation has been associated with fibrosis in the lung, kidney, and skin, it is unknown whether hedgehog signaling activation is the cause or the consequence of fibrosis. We hypothesize that activation of hedgehog signaling is sufficient to drive fibrosis in mouse models.ResultsIn this study, we provide direct evidence to show that activation of Hh signaling via expression of activated smoothened, SmoM2, is sufficient to induce fibrosis in the vasculature and aortic valves. We showed that activated SmoM2 -induced fibrosis is associated with abnormal function of aortic valves and heart. The relevance of this mouse model to human health is reflected in our findings that elevated GLI expression is detected in 6 out of 11 aortic valves from patients with fibrotic aortic valves.ConclusionsOur data show that activating hedgehog signaling is sufficient to drive fibrosis in mice, and this mouse model is relevant to human aortic valve stenosis.

Deficiency of HtrA3 Attenuates Bleomycin-Induced Pulmonary Fibrosis Via TGF-β1/Smad Signaling Pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by excessive extracellular matrix deposition. No effective treatments are currently available for IPF. High-temperature requirement A3 (HtrA3) suppresses tumor development by antagonizing transforming growth factor β (TGF-β) signaling; however, little is known about the role of HtrA3 in IPF. This study investigated the role of HtrA3 in IPF and underlying mechanisms. Lung tissues were collected from patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis, and HtrA3 expression was measured in tissue samples. Then, HtrA3 gene knockout mice were treated with BLM to induce pulmonary fibrosis and explore the effects and underlying mechanism of HtrA3 on pulmonary fibrosis. HtrA3 was up-regulated in the lung tissues of patients with IPF and the pulmonary fibrotic mouse model compared to corresponding control groups. HtrA3 knockout decreased pulmonary fibrosis-related protein expression, alleviated the symptoms of pulmonary fibrosis, and inhibited epithelial-mesenchymal transition (EMT) in BLM-induced lung tissue compared with BLM-induced wild-type mice. The TGF-β1/Smad signaling pathway was activated in fibrotic lung tissue, whereas HtrA3 knockout inhibited this pathway. The expression level of HtrA3 is increased in fibrotic lungs. HtrA3 knockout alleviates the symptoms of pulmonary fibrosis probably via the TGF-β1/Smad signaling pathway. Therefore, HtrA3 inhibition is a potential therapeutic target for pulmonary fibrosis.

Docosahexaenoic acid alleviates the excessive degradation of extracellular matrix in the nucleus pulposus by reducing the content of lncRNA NEAT1 to prevent the progression of intervertebral disc degeneration.

The pathogenesis of intervertebral disc degeneration (IVDD), as a multifactorial disease, has not been fully elucidated. However, damage to the stress-bearing system in the intervertebral disc (IVD) mediated by the excessive decomposition of extracellular matrix (ECM) in nucleus pulposus (NP) cells caused by local stimulation is widely considered the core pathological process underlying IVDD. Docosahexaenoic acid (DHA) plays a protective role in various chronic diseases. However, whether it can have such effects in IVDD has not been clearly reported. In recent years, in-depth research on the role of long non-coding RNA (lncRNA) nuclear-enriched transcript 1 (NEAT1) in various diseases has continuously emerged, but such research in the field of IVD is not sufficient. In this study, tert-butyl hydroperoxide (TBHP) was used to induce oxidative stress in human NP cells and construct a cell model of excessive ECM decomposition in vitro. A plasmid over-expressing lncRNA NEAT1 was introduced into human NP cells to establish an NP cell model. For this specific experiment, Cell Counting Kit 8 was used to explore the timing and concentration of DHA and TBHP activity. A common gene chip platform was also used to select potential lncRNAs. Western blot and immunofluorescence assays were used to detect the expression of ECM-related proteins in NP cells in each group. Quantitative real-time polymerase chain reaction was used to detect the expression of lncRNA NEAT1 in NP cells in each group. On this basis, we proved that DHA alleviates excessive degradation of the ECM in NP cells in response to oxidative stress by reducing the content of lncRNA NEAT1. In conclusion, our study reveals the mechanism through which DHA relieves excessive ECM decomposition in NP cells and provides a potential new idea for the treatment of IVDD in clinical practice.

Tyrosine kinase receptor B attenuates liver fibrosis by inhibiting TGF-β/SMAD signaling.

Liver fibrosis is a leading indicator for increased mortality and long-term comorbidity in NASH. Activation of HSCs and excessive extracellular matrix production are the hallmarks of liver fibrogenesis. Tyrosine kinase receptor (TrkB) is a multifunctional receptor that participates in neurodegenerative disorders. However, paucity of literature is available about TrkB function in liver fibrosis. Herein, the regulatory network and therapeutic potential of TrkB were explored in the progression of hepatic fibrosis. The protein level of TrkB was decreased in mouse models of CDAHFD feeding or carbon tetrachloride-induced hepatic fibrosis. TrkB suppressed TGF-β-stimulated proliferation and activation of HSCs in 3-dimensional liver spheroids and significantly repressed TGF-β/SMAD signaling pathway either in HSCs or in hepatocytes. The cytokine, TGF-β, boosted Nedd4 family interacting protein-1 (Ndfip1) expression, promoting the ubiquitination and degradation of TrkB through E3 ligase Nedd4-2. Moreover, carbon tetrachloride intoxication-induced hepatic fibrosis in mouse models was reduced by adeno-associated virus vector serotype 6 (AAV6)-mediated TrkB overexpression in HSCs. In addition, in murine models of CDAHFD feeding and Gubra-Amylin NASH (GAN), fibrogenesis was reduced by adeno-associated virus vector serotype 8 (AAV8)-mediated TrkB overexpression in hepatocytes. TGF-β stimulated TrkB degradation through E3 ligase Nedd4-2 in HSCs. TrkB overexpression inhibited the activation of TGF-β/SMAD signaling and alleviated the hepatic fibrosis both in vitro and in vivo . These findings demonstrate that TrkB could be a significant suppressor of hepatic fibrosis and confer a potential therapeutic target in hepatic fibrosis.

Upregulation of miR-200a improves ureteral obstruction-induced renal fibrosis via GAB1/Wnt/β-catenin signaling

BackgroundRenal fibrosis is a basic pathological change of almost all chronic kidney disorders. Epithelial–mesenchymal transition (EMT) and excessive extracellular matrix (ECM) accumulation play a crucial role in the process of fibrosis. MethodsWestern blot and qRT-PCR were accomplished to analyze the expression levels of target proteins and genes, respectively. The fibrotic levels in the renal tissues of rats were confirmed utilizing Masson staining. Expression of ECM-related α-SMA in the renal tissues was determined by immunohistochemistry assay. The combination of GRB2 associated binding protein 1 (GAB1) and miR-200a was ensured by starBase database and luciferase reporter assay. ResultsOur data uncovered that miR-200a was downregulated, but GAB1 was upregulated in the renal tissues of the rat experienced unilateral ureteral obstruction (UUO). Overexpression of miR-200a improved tissues fibrosis, suppressed GAB1 expression and ECM deposition, and inactivated Wnt/β-catenin in UUO rats. Moreover, miR-200a expression was inhibited, while GAB1 expression was facilitated in the TGF-β1-induced HK-2 cells. In TGF-β1-induced HK-2 cells, miR-200a overexpression inhibited GAB1 expression, also declined ECM-related proteins and mesenchymal markers expression. Oppositely, miR-200a overexpression facilitated epithelial marker expression in the TGF-β1-induced HK-2 cells. Next, the data revealed that miR-200a inhibited GAB1 expression through binding to the mRNA 3′-UTR of GAB1. Increasing of GAB1 reversed the regulation of miR-200a to GAB1 expression, Wnt/β-catenin signaling activation, EMT and ECM accumulation. ConclusionOverall, miR-200a increasing improved renal fibrosis through attenuating EMT and ECM accumulation by limiting Wnt/β-catenin signaling via sponging GAB1, indicating miR-200a may be a promising objective for renal disease therapy.

Macrophage-derived GPNMB trapped by fibrotic extracellular matrix promotes pulmonary fibrosis

Pulmonary fibrosis (PF) is a form of progressive lung disease characterized by chronic inflammation and excessive extracellular matrix (ECM) deposition. However, the protein changes in fibrotic ECM during PF and their contribution to fibrosis progression are unclear. Here we show that changes in expression of ECM components and ECM remodeling had occurred in silica-instilled mice. The macrophage-derived glycoprotein nonmetastatic melanoma protein B (GPNMB) captured by fibrotic ECM may activate resident normal fibroblasts around the fibrotic foci. Functional experiments demonstrated the activation of fibroblasts in fibrotic ECM, which was alleviated by GPNMB-neutralizing antibodies or macrophage deletion in the ECM of silica-instilled mice. Moreover, the Serpinb2 expression level was increased in fibroblasts in fibrotic ECM, and the expression of CD44 was increased in silica-instilled mice. In conclusion, macrophage-derived GPNMB is trapped by fibrotic ECM during transport and may activate fibroblasts via the CD44/Serpinb2 pathway, thus leading to the further development of fibrosis.

P118 Induction of ETS1 and senescence associated secretory phenotype in human intestinal fibroblasts

Abstract Background Fibrosis is a complication commonly present in Crohn’s disease (CD) patients with a structuring (B2) or penetrating (B3) behaviour, with no available treatment. This process is characterized by an excessive extracellular matrix deposition, mainly associated with a dysregulated function of myofibroblasts. We analyse here, the expression of markers of senescence in human intestinal fibroblasts. Methods Human small intestinal fibroblasts (HSIF; P10760, Innoprot, Spain) were treated during 48h with 2µg/mL of DLL4, 20ng/ml of TNFα, 2ng/ml of IL-1β, 5ng/ml of TGFβ1, and 100ng/ml of. In some cases, fibroblasts were treated with 20nM of siRNA to ETS1 gene (siETS21) or negative control (NC). Gene expression profiles were analysed by RT-qPCR. Results Treatment of fibroblasts with IL-1β significantly increased the mRNA expression of a) different senescence-associated secretory phenotype (SASP) factors (IL-1α, IL-1β, IL-8, and Serpine1); b) a metalloprotease ADAM12 and the chitinase CHI3L1 and c) the transcription factor related to senescence ETS proto-oncogene 1 (ETS1), all compared with vehicle treatment. Treatment with PDGF significantly increased the mRNA expression of ADAM12, ETS1, PTEN and the transcription factor, E2F5, while it induced a non-significant increase in the expression of senescence markers such as P16, P21, P53 and MCL1. Treatment with TGFβ1 did not significantly alter the expression of markers reported above and it only significantly increased the expression of IGFBP3 gene, another SASP factor. Both DLL4 and TNFα failed to significantly modify the expression of any of the above markers. Finally, a cellular model of depletion of ETS1 showed that siETS1 fibroblasts in basal conditions exhibited decreased levels of IL-8 and BCL2. Conclusion In human primary intestinal fibroblasts, treatment with IL1b increased the expression of senescence associated secretory phenotype while treatment with PDGF increased the expression of markers involved in cell cycle arrest. The induction of ETS1 by both treatments and the involvement of this transcription factor regulating gene expression in basal conditions, suggest a relevant role for ETS1 in the activation of intestinal fibroblasts

Ciclopirox Inhibition of eIF5A Hypusination Attenuates Fibroblast Activation and Cardiac Fibrosis.

Cardiac fibrosis is a primary contributor to heart failure (HF), and is considered to be a targetable process for HF therapy. Cardiac fibroblast (CF) activation accompanied by excessive extracellular matrix (ECM) production is central to the initiation and maintenance of fibrotic scarring in cardiac fibrosis. However, therapeutic compounds targeting CF activation remain limited in treating cardiac fibrosis. Eukaryotic translation initiation factor 5A (eIF5A), upon being hypusinated, is essential for the translation elongation of proline-codon rich mRNAs. In this study, we found that increased hypusinated eIF5A protein levels were associated with cardiac fibrosis and heart dysfunction in myocardial infarction (MI) mouse models. Ciclopirox (CPX), an FDA-approved antifungal drug, inhibits the deoxyhypusine hydroxylase (DOHH) enzyme required for eIF5A hypusination. Results from preventive and reversal mouse models suggest that CPX treatment significantly reduced MI-driven cardiac fibrosis and improved cardiac function. In vitro studies of isolated mouse primary CFs revealed that inhibition of eIF5A hypusination using CPX significantly abolished TGFβ induced CF proliferation, activation, and collagen expression. Proteomic analysis from mouse CFs reveals that CPX downregulates the expression of proline-rich proteins that are enriched in extracellular matrix and cell adhesion pathways. Our findings are relevant to human heart disease, as increased hypusinated eIF5A levels were observed in heart samples of ischemic heart failure patients compared to healthy subjects. Together, these results suggest that CPX can be repurposed to treat cardiac fibrosis and ischemic heart failure.

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.

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.

Mechanical stress regulates the mechanotransduction and metabolism of cardiac fibroblasts in fibrotic cardiac diseases

Fibrotic cardiac diseases are characterized by myocardial fibrosis that results in maladaptive cardiac remodeling. Cardiac fibroblasts (CFs) are the main cell type responsible for fibrosis. In response to stress or injury, intrinsic CFs develop into myofibroblasts and produce excess extracellular matrix (ECM) proteins. Myofibroblasts are mechanosensitive cells that can detect changes in tissue stiffness and respond accordingly. Previous studies have revealed that some mechanical stimuli control fibroblast behaviors, including ECM formation, cell migration, and other phenotypic traits. Further, metabolic alteration is reported to regulate fibrotic signaling cascades, such as the transforming growth factor-β pathway and ECM deposition. However, the relationship between metabolic changes and mechanical stress during fibroblast-to-myofibroblast transition remains unclear. This review aims to elaborate on the crosstalk between mechanical stress and metabolic changes during the pathological transition of cardiac fibroblasts.

The uPA/uPAR System Orchestrates the Inflammatory Response, Vascular Homeostasis, and Immune System in Fibrosis Progression.

Fibrotic diseases, such as systemic sclerosis (SSc), idiopathic pulmonary fibrosis, renal fibrosis and liver cirrhosis are characterized by tissue overgrowth due to excessive extracellular matrix (ECM) deposition. Fibrosis progression is caused by ECM overproduction and the inhibition of ECM degradation due to several events, including inflammation, vascular endothelial dysfunction, and immune abnormalities. Recently, it has been reported that urokinase plasminogen activator (uPA) and its receptor (uPAR), known to be fibrinolytic factors, orchestrate the inflammatory response, vascular homeostasis, and immune homeostasis system. The uPA/uPAR system may show promise as a potential therapeutic target for fibrotic diseases. This review considers the role of the uPA/uPAR system in the progression of fibrotic diseases.

Phillygenin inhibited M1 macrophage polarization and reduced hepatic stellate cell activation by inhibiting macrophage exosomal miR-125b-5p

Liver fibrosis (LF) is an important stage in chronic liver disease development, characterized by hepatic stellate cell (HSC) activation and excessive extracellular matrix deposition. Phillygenin (PHI), an active component in the traditional Chinese medicine Forsythiae Fructus with a significant anti-inflammatory effect, has been proved to inhibit HSC activation. Macrophages can polarize to pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype, participating in LF development. Currently, Forsythiae Fructus and its many components have been proved to inhibit the inflammatory activation of macrophages. However, there is no direct evidence that PHI can regulate macrophage polarization, and the relationship between macrophage polarization and the anti-LF effect of PHI has not been studied. In this study, we found that PHI inhibited the co-expression of CD80 and CD86, and inhibited the mRNA expression and protein secretion of related inflammatory cytokines in RAW264.7 cells. For mechanism, PHI was found to inhibit the JAK1/JAK2-STAT1 and Notch1 signaling pathways. Subsequently, mHSCs were co-cultured with the conditioned media or exosomes from macrophages with different treatments. It was found that the conditioned media and exosomes from PHI-treated macrophages inhibited the expression of MMP2, TIMP1, TGF-β, α-SMA, COL1 and NF-κB in mHSCs. Moreover, through bioinformatic analysis and cell transfection, we confirmed that PHI reduced HSC activation by inhibiting the overexpression of miR-125b-5p in M1 macrophage-derived exosomes and restoring Stard13 expression in mHSCs. On the whole, PHI could inhibit M1 macrophage polarization by suppressing the JAK1/JAK2-STAT1 and Notch1 signaling pathways, and reduce HSC activation by inhibiting macrophage exosomal miR-125b-5p targeting Stard13. Data AvailabilityThe raw data supporting the conclusions of this study are available in the article/Supplementary figures, and can be obtained from the first or corresponding author.

Dysregulated B cell function and disease pathogenesis in systemic sclerosis.

Systemic sclerosis (SSc) is a complex, immune-mediated rheumatic disease characterised by excessive extracellular matrix deposition in the skin and internal organs. B cell infiltration into lesional sites such as the alveolar interstitium and small blood vessels, alongside the production of defined clinically relevant autoantibodies indicates that B cells play a fundamental role in the pathogenesis and development of SSc. This is supported by B cell and fibroblast coculture experiments revealing that B cells directly enhance collagen and extracellular matrix synthesis in fibroblasts. In addition, B cells from SSc patients produce large amounts of profibrotic cytokines such as IL-6 and TGF-β, which interact with other immune and endothelial cells, promoting the profibrotic loop. Furthermore, total B cell counts are increased in SSc patients compared with healthy donors and specific differences can be found in the content of naïve, memory, transitional and regulatory B cell compartments. B cells from SSc patients also show differential expression of activation markers such as CD19 which may shape interactions with other immune mediators such as T follicular helper cells and dendritic cells. The key role of B cells in SSc is further supported by the therapeutic benefit of B cell depletion with rituximab in some patients. It is notable also that B cell signaling is impaired in SSc patients, and this could underpin the failure to induce tolerance in B cells as has been shown in murine models of scleroderma.

Lipid nanoparticle-mediated mRNA delivery in lung fibrosis

mRNA delivery enables the specific synthesis of proteins with therapeutic potential, representing a powerful strategy in diseases lacking efficacious pharmacotherapies. Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by excessive extracellular matrix (ECM) deposition and subsequent alveolar remodeling. Alveolar epithelial type 2 cells (AEC2) and fibroblasts represent important targets in IPF given their role in initiating and driving aberrant wound healing responses that lead to excessive ECM deposition. Our objective was to examine a lipid nanoparticle (LNP)-based mRNA construct as a viable strategy to target alveolar epithelial cells and fibroblasts in IPF. mRNA-containing LNPs measuring ∼34 nm had high encapsulation efficiency, protected mRNA from degradation, and exhibited sustained release kinetics. eGFP mRNA LNP transfection in human primary cells proved dose- and time-dependent in vitro. In a bleomycin mouse model of lung fibrosis, luciferase mRNA LNPs administered intratracheally led to site-specific lung accumulation. Importantly, bioluminescence signal was detected in lungs as early as 2 h after delivery, with signal still evident at 48 h. Of note, LNPs were found associated with AEC2 and fibroblasts in vivo. Findings highlight the potential for pulmonary delivery of mRNA in IPF, opening therapeutic avenues aimed at halting and potentially reversing disease progression.

High Throughput Confined Migration Microfluidic Device for Drug Screening.

Cancer metastasis is the major cause of cancer-related death. Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors, creating confined spaces for tumor cell migration and metastasis. Confined migration is involved in all metastasis steps. However, confined and unconfined migration inhibitors are different and drugs available to inhibit confined migration are rare. The main challenges are the modeling of confined migration, the suffering of low throughput, and others. Microfluidic device has the advantage to reduce reagent consumption and enhance throughput. Here, a microfluidic chip that can achieve multi-function drug screening against the collective migration of cancer cells under confined environment is designed. This device is applied to screen out effective drugs on confined migration among a novel mechanoreceptors compound library (166 compounds) in hepatocellular carcinoma, non-small lung cancer, breast cancer, and pancreatic ductal adenocarcinoma cells. Three compounds that can significantly inhibit confined migration in pan-cancer: mitochonic acid 5 (MA-5), SB-705498, and diphenyleneiodonium chloride are found. Finally, it is elucidated that these drugs targeted mitochondria, actin polymerization, and cell viability, respectively. In sum, a high-throughput microfluidic platform for screening drugs targeting confined migration is established and three novel inhibitors of confined migration in multiple cancer types are identified.