Antifibrotic Target Research Articles

Calcipotriol abrogates TGF-β1/pSmad3-mediated collagen 1 synthesis in pancreatic stellate cells by downregulating RUNX1

RUNX1 with CBFβ functions as an activator or repressor of critical mediators regulating cellular function. The aims of this study were to clarify the role of RUNX1 on regulating TGF-β1-induced COL1 synthesis and the mechanism of calcipotriol (Cal) on antagonizing COL1 synthesis in PSCs. RT-qPCR and Western Blot for determining the mRNAs and proteins of RUNX1 and COL1A1/1A2 in rat PSC line (RP-2 cell). Luciferase activities driven by RUNX1 or COL1A1 or COL1A2 promoter, co-immunoprecipitation and immunoblotting for pSmad3/RUNX1 or CBFβ/RUNX1, and knockdown or upregulation of Smad3 and RUNX1 were used. RUNX1 production was regulated by TGF-β1/pSmad3 signaling pathway in RP-2 cells. RUNX1 formed a coactivator with CBFβ in TGF-β1-treated RP-2 cells to regulate the transcriptions of COL1A1/1A2 mRNAs under a fashion of pSmad3/RUNX1/CBFβ complex. However, Cal effectively abrogated the levels of COL1A1/1A2 transcripts in TGF-β1-treated RP-2 cells by downregulating RUNX1 production and hindering the formation of pSmad3/RUNX1/CBFβ complexes. This study suggests that RUNX1 may be a promising antifibrotic target for the treatment of chronic pancreatitis.

Interference with mitochondrial function as part of the antifibrogenic effect of Rilpivirine: A step towards novel targets in hepatic stellate cell activation

Activated hepatic stellate cells (aHSCs), the main perpetrators of liver fibrosis, are a promising therapeutic target in the treatment of chronic liver disease. During liver injury, HSCs transcend from a quiescent to a fibrotic phenotype, a process which involves major metabolic reprogramming with altered mitochondrial function. The antiretroviral drug Rilpivirine (RPV) has demonstrated a hepatoprotective and specifically antifibrotic effect in several animal models of chronic liver injury, as well as in vitro. Herein, we use HSCs activated with the profibrogenic cytokine TGF-β to explore whether mitochondrial function is implicated in this effect. The mitochondrial bioenergetic profile, morphology and dynamics of TGF-β-treated cells (48 h) were altered and these effects were prevented by co-treatment with clinically relevant concentrations of RPV. A MitoStress Test (Seahorse Analyzer) revealed that TGF-β increased both oxygen consumption rate (basal respiration, maximal respiration and spare respiratory capacity) and extracellular acidification rate (indicative of increased glycolysis). Cells exposed to TGF-β also displayed diminished mitochondrial membrane potential and enhanced mitochondrial fission. All of these effects were rescued with RPV. RNA sequencing analysis of cells exposed to TGF-β revealed the presence of 338 differentially expressed genes that encode mitochondrial proteins (mito-DEGs), of which 139 and 199 were significantly up- and down-regulated (adjusted p<0.05). This alteration in 15 (10.79 %) and 31 (22.03 %) of the up-regulated and 16 (8.04 %) and 49 (24.62 %) of the down-regulated mitoDEGs was prevented with co-exposure to RPV 4μM or 8μM, respectively. In conclusion, alterations in mitochondrial function are implicated in the antifibrogenic action of RPV, pointing to potential novel antifibrotic targets.

Hepatitis C virus NS5A and core protein induce fibrosis-related genes regulation on Huh7 cells through activation of LX2 cells

Introduction and ObjectivesLiver fibrosis remains a complication derived from a chronic Hepatitis C Virus (HCV) infection even when it is resolved, and no liver antifibrotic drug has been approved. Molecular mechanisms on hepatocytes and activation of hepatic stellate cells (HSCs) play a central role in liver fibrogenesis. To elucidate molecular mechanisms, it is important to analyze pathway regulation during HSC activation and HCV infection. Materials and MethodsWe evaluate the fibrosis-associated molecular mechanisms during a co-culture of human HSCs (LX2), with human hepatocytes (Huh7) that express HCV NS5A or Core protein. We evaluated LX2 activation induced by HCV NS5A or Core expression in Huh7 cells during co-culture. We determined a fibrosis-associated gene expression profile in Huh7 that expresses NS5A or Core proteins during the co-culture with LX2. ResultsWe observed that NS5A induced 8.3-, 6.7- and 4-fold changes and that Core induced 6.5-, 1.8-, and 6.2-fold changes in the collagen1, TGFβ1, and timp1 gene expression, respectively, in LX2 co-cultured with transfected Huh7. In addition, NS5A induced the expression of 30 genes while Core induced 41 genes and reduced the expression of 30 genes related to fibrosis in Huh7 cells during the co-culture with LX2, compared to control. The molecular pathways enriched from the gene expression profile were involved in TGFB signaling and the organization of extracellular matrix. ConclusionsWe demonstrated that HCV NS5A and Core protein expression regulate LX2 activation. NS5A and Core-induced LX2 activation, in turn, regulates diverse fibrosis-related gene expression at different levels in Huh7, which can be further analyzed as potential antifibrotic targets during HCV infection.

A small-molecule TNIK inhibitor targets fibrosis in preclinical and clinical models.

Idiopathic pulmonary fibrosis (IPF) is an aggressive interstitial lung disease with a high mortality rate. Putative drug targets in IPF have failed to translate into effective therapies at the clinical level. We identify TRAF2- and NCK-interacting kinase (TNIK) as an anti-fibrotic target using a predictive artificial intelligence (AI) approach. Using AI-driven methodology, we generated INS018_055, a small-molecule TNIK inhibitor, which exhibits desirable drug-like properties and anti-fibrotic activity across different organs in vivo through oral, inhaled or topical administration. INS018_055 possesses anti-inflammatory effects in addition to its anti-fibrotic profile, validated in multiple in vivo studies. Its safety and tolerability as well as pharmacokinetics were validated in a randomized, double-blinded, placebo-controlled phase I clinical trial (NCT05154240) involving 78 healthy participants. A separate phase I trial in China, CTR20221542, also demonstrated comparable safety and pharmacokinetic profiles. This work was completed in roughly 18 months from target discovery to preclinical candidate nomination and demonstrates the capabilities of our generative AI-driven drug-discovery pipeline.

Search progress of pyruvate kinase M2 (PKM2) in organ fibrosis.

Fibrosis is characterized by abnormal deposition of the extracellular matrix (ECM), leading to organ structural remodeling and loss of function. The principal cellular effector in fibrosis is activated myofibroblasts, which serve as the main source of matrix proteins. Metabolic reprogramming, transitioning from mitochondrial oxidative phosphorylation to aerobic glycolysis, is widely observed in rapidly dividing cells such as tumor cells and activated myofibroblasts and is increasingly recognized as a fundamental pathogenic basis in organ fibrosis. Targeting metabolism represents a promising strategy to mitigate fibrosis. PKM2, a key enzyme in glycolysis, plays a pivotal role in metabolic reprogramming through allosteric regulation, impacting both metabolic and non-metabolic pathways. Therefore, metabolic reprogramming induced by PKM2 activation is involved in the occurrence and development of fibrosis in various organs. A comprehensive understanding of the role of PKM2 in fibrotic diseases is crucial for seeking new anti-fibrotic therapeutic targets. In this context, we summarize PKM2's role in glycolysis, mediating the intricate mechanisms underlying fibrosis in multiple organs, and discuss the potential value of PKM2 inhibitors and allosteric activators in future clinical treatments, aiming to identify novel therapeutic targets for proliferative fibrotic diseases.

Potential Rheumatoid Arthritis-Associated Interstitial Lung Disease Treatment and Computational Approach for Future Drug Development.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by swelling in at least one joint. Owing to an overactive immune response, extra-articular manifestations are observed in certain cases, with interstitial lung disease (ILD) being the most common. Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is characterized by chronic inflammation of the interstitial space, which causes fibrosis and the scarring of lung tissue. Controlling inflammation and pulmonary fibrosis in RA-ILD is important because they are associated with high morbidity and mortality. Pirfenidone and nintedanib are specific drugs against idiopathic pulmonary fibrosis and showed efficacy against RA-ILD in several clinical trials. Immunosuppressants and disease-modifying antirheumatic drugs (DMARDs) with anti-fibrotic effects have also been used to treat RA-ILD. Immunosuppressants moderate the overexpression of cytokines and immune cells to reduce pulmonary damage and slow the progression of fibrosis. DMARDs with mild anti-fibrotic effects target specific fibrotic pathways to regulate fibrogenic cellular activity, extracellular matrix homeostasis, and oxidative stress levels. Therefore, specific medications are required to effectively treat RA-ILD. In this review, the commonly used RA-ILD treatments are discussed based on their molecular mechanisms and clinical trial results. In addition, a computational approach is proposed to develop specific drugs for RA-ILD.

P117 Transglutaminase 2 is elevated in Crohn’s disease associated strictures and exerts profibrotic activities in myofibroblasts and experimental intestinal fibrosis

Abstract Background Intestinal fibrosis is a significant clinical problem in Inflammatory bowel disease (IBD). This may lead to intestinal strictures induced by excessive accumulation of extracellular matrix (ECM) produced by human intestinal myofibroblasts (HIMF). Transglutaminase (TG)2 covalently cross-links ECM-associated proteins, increases ECM stiffness and has direct pro-fibrotic effects. We investigated the role of TG2 in intestinal fibrosis in human tissues, HIMF and transgenic mouse models. Methods TG2 was detected in human tissues from Crohn’s disease (CD) strictures (CDs; n=10), CD non-strictured (CDns; n=20), ulcerative colitis (UC; n=10) and non-IBD controls (NL; n=14) by immunofluorescence (IF), qPCR, in-situ hybridization (ISH), and biotinylated cadaverin incorporation (BCI). Relative contribution of TG2 was measured by BCI using a TG2-selective blocking antibody (TAb). The cellular source of TG2 was evaluated by IF. HIMF ECM production was measured using a novel ECM deposition assay and TG2-inhibitors. HIMF were exposed to anti-TG2 antibody or isotype control and bulk RNA-sequencing (RNAseq) analysis was performed. Tamoxifen-inducible global Cre/TG2 floxed conditional KO (cKO) mice were used in the DSS and TNBS fibrosis models. Results Total TG activity was increased in CDs and UC vs. NL in the submucosa (SM) and muscularis propria (MP; P≤ 0.04). Strikingly 90-99% of TG activity were attributable to TG2 in all groups. TG2 activity correlated with fibrosis scores (P≤0.02), but not inflammation scores. TG2 amount was elevated in CDs patients vs. NL in qPCR and ISH (P≤0.05). IF revealed HIMF as the major source of TG2 in the intestine as indicated by colocalization with alpha-SMA. HIMF, invitro, produced 82–97% of active TG2. Inhibition of TG2 by cell-permeable inhibitor or Tab reduced fibronectin (FN) and collagen (COL)1 and 3 deposition by HIMF. RNAseq showed TAb-treated cells expressed 432 downregulated and 402 upregulated genes. Functional enrichment analysis showed pathways associated with fibroblast activation/proliferation, ECM, and fibrosis-associated signaling (e.g. AKT) pathways to be downregulated in TAb-treated HIMF. Associated genes included nuclear protein (NUPR1) and sphingolipid signaling pathway proteins (SMPD1 and CTSD), which may be potential targets for TG2-mediated activity in HIMF. Deletion of TGM2 prior to inducing murine DSS or TNBS experimental fibrosis did not lead to any difference in inflammation, but decreased fibrosis (picrosirius red intensity, COL1/FN expression, wall thickness) in TG2 cKO mice vs. wild type (P≤0.05). Conclusion TG2 is involved in inflammation-independent progression of intestinal fibrosis and may represent a future anti-fibrotic target.

The effect of TG2-inhibitory monoclonal antibody zampilimab on tissue fibrosis in human in vitro and primate in vivo models of chronic kidney disease.

Fibrotic remodeling is the primary driver of functional loss in chronic kidney disease, with no specific anti-fibrotic agent available for clinical use. Transglutaminase 2 (TG2), a wound response enzyme that irreversibly crosslinks extracellular matrix proteins causing dysregulation of extracellular matrix turnover, is a well-characterized anti-fibrotic target in the kidney. We describe the humanization and characterization of two anti-TG2 monoclonal antibodies (zampilimab [hDC1/UCB7858] and BB7) that inhibit crosslinking by TG2 in human in vitro and rabbit/cynomolgus monkey in vivo models of chronic kidney disease. Determination of zampilimab half-maximal inhibitory concentration (IC50) against recombinant human TG2 was undertaken using the KxD assay and determination of dissociation constant (Kd) by surface plasmon resonance. Efficacy in vitro was established using a primary human renal epithelial cell model of tubulointerstitial fibrosis, to assess mature deposited extracellular matrix proteins. Proof of concept in vivo used a cynomolgus monkey unilateral ureteral obstruction model of chronic kidney disease. Zampilimab inhibited TG2 crosslinking transamidation activity with an IC50 of 0.25 nM and Kd of <50 pM. In cell culture, zampilimab inhibited extracellular TG2 activity (IC50 119 nM) and dramatically reduced transforming growth factor-β1-driven accumulation of multiple extracellular matrix proteins including collagens I, III, IV, V, and fibronectin. Intravenous administration of BB7 in rabbits resulted in a 68% reduction in fibrotic index at Day 25 post-unilateral ureteral obstruction. Weekly intravenous administration of zampilimab in cynomolgus monkeys with unilateral ureteral obstruction reduced fibrosis at 4 weeks by >50%, with no safety signals. Our data support the clinical investigation of zampilimab for the treatment of kidney fibrosis.

SLIT3-mediated fibroblast signaling: a promising target for antifibrotic therapies.

The SLIT family (SLIT1-3) of highly conserved glycoproteins was originally identified as ligands for the Roundabout (ROBO) family of single-pass transmembrane receptors, serving to provide repulsive axon guidance cues in the nervous system. Intriguingly, studies involving SLIT3 mutant mice suggest that SLIT3 might have crucial biological functions outside the neural context. Although these mutant mice display no noticeable neurological abnormalities, they present pronounced connective tissue defects, including congenital central diaphragmatic hernia, membranous ventricular septal defect, and osteopenia. We recently hypothesized that the phenotype observed in SLIT3-deficient mice may be tied to abnormalities in fibrillar collagen-rich connective tissue. Further research by our group indicates that both SLIT3 and its primary receptor, ROBO1, are expressed in fibrillar collagen-producing cells across various nonneural tissues. Global and constitutive SLIT3 deficiency not only reduces the synthesis and content of fibrillar collagen in various organs but also alleviates pressure overload-induced fibrosis in both the left and right ventricles. This review delves into the known phenotypes of SLIT3 mutants and the debated role of SLIT3 in vasculature and bone. Present evidence hints at SLIT3 acting as an autocrine regulator of fibrillar collagen synthesis, suggesting it as a potential antifibrotic treatment. However, the precise pathway and mechanisms through which SLIT3 regulates fibrillar collagen synthesis remain uncertain, presenting an intriguing avenue for future research.

Found in translation-Fibrosis in metabolic dysfunction-associated steatohepatitis (MASH).

Metabolic dysfunction-associated steatohepatitis (MASH) is a severe form of liver disease that poses a global health threat because of its potential to progress to advanced fibrosis, leading to cirrhosis and liver cancer. Recent advances in single-cell methodologies, refined disease models, and genetic and epigenetic insights have provided a nuanced understanding of MASH fibrogenesis, with substantial cellular heterogeneity in MASH livers providing potentially targetable cell-cell interactions and behavior. Unlike fibrogenesis, mechanisms underlying fibrosis regression in MASH are still inadequately understood, although antifibrotic targets have been recently identified. A refined antifibrotic treatment framework could lead to noninvasive assessment and targeted therapies that preserve hepatocellular function and restore the liver's architectural integrity.

UPLC-Q-TOF-MS based investigation into the bioactive compounds and molecular mechanisms of Lamiophlomis Herba against hepatic fibrosis

BackgroundLamiophlomis Herba (LH) is a valuable traditional medicinal plant found on the Qinghai-Tibetan Plateau that promotes blood circulation, removes blood stasis, and has antibacterial and anti-inflammatory properties. The main components of LH are iridoid glycosides, phenethyl alcohol glycosides, flavonoids, and polysaccharides. PurposeTo investigate the mechanism of the anti-liver fibrosis effects of LH and screen for its bioactive compounds. Study designScreening LH marker components and validating the LH anti-liver fibrosis mechanism. MethodsThe active ingredients of LH were identified using UPLC-Q-TOF-MS, and HotMap combined with principal components analysis (PCA) was used to screen for marker components. Network pharmacology and molecular docking techniques were used to predict the potential anti-fibrotic targets of LH. Immunofluorescence, enzyme-linked immunosorbent assay (ELISA), real-time PCR (RT-PCR), and western blotting were used for experimental validation and mechanistic studies. ResultsFifteen compounds that actively contributed to the cluster were identified as marker compounds. Acteoside, 8-O-acetyl shanzhiside methyl ester (8-O-ASME), Luteolin, Shanzhiside Methyl ester (SME), Loganin, Loganate were the main active components. Network pharmacology and molecular docking studies have shown that LH might improve liver fibrosis, inflammation, and oxidative stress, which might be related to key targets such as PTGS2, MAPK, EGFR, AKT1, SRC, Fn1, Col3a1, Col1a1, and PC-III. The results of ELISA, RT-PCR and western blot experiments showed that Acteoside, 8-O-ASME, Luteolin, SME, Loganin, Loganate, and the LH group could reduce the levels of fibronectin, Col1a1, Col3a1, α-SMA, Col-Ⅳ, LN, and PC-Ⅲ. ConclusionLH improves liver fibrosis induced by HSC-T6 cells and inhibits the deposition of extracellular matrix (ECM) in hepatocytes, resulting in a decrease in the degree of liver fibrosis and a good anti-liver fibrosis effect.

Salvianolic acid B inhibits hepatic stellate cell activation and liver fibrosis by targeting PDGFRβ.

Liver fibrosis is a reversible pathological process and a wound healing response to liver injury. As an early stage of various liver diseases, liver fibrosis can develop into cirrhosis, liver failure, and even liver cancer if not controlled in time. Salvia miltiorrhiza is a medicinal plant with hepatoprotective effects. Salvianolic acid B (Sal B) is the representative component of S. miltiorrhiza. Many studies have reported the anti-liver fibrosis effects and mechanisms of Sal B. However, the direct anti-fibrotic targets of Sal B have not yet been reported. Platelet-derived growth factor receptor β (PDGFRβ) is one of the most classical targets in liver fibrosis, which is closely related to hepatic stellate cells (HSCs) activated. Previously, we established and applied a PDGFRβ affinity chromatography model, and found that Sal B binds well to PDGFRβ. Therefore, this study aimed to investigate the direct targets of Sal B against liver fibrosis. We confirmed the binding ability of Sal B to PDGFRβ by molecular docking and a surface plasmon resonance biosensor. Our findings indicated that Sal B targeted PDGFRβ to inhibit the activation, migration and proliferation of HSCs and suppressed the PDGF-BB-induced PDGFRβ signaling pathway. Annexin V-FITC/PI assay showed that Sal B reversed the PDGF-BB-induced decrease in HSC apoptosis rate. In the mouse liver fibrosis model, Sal B inhibited the PDGFRβ signaling pathway, HSC activation and reduced inflammatory response, ultimately improved CCl4-induced liver fibrosis. In summary, the direct anti-fibrotic targets of Sal B may be PDGFRβ, and this study clarified the anti-liver fibrosis effects and mechanism of Sal B.

ATRA ameliorates fibrosis by suppressing the pro-fibrotic molecule Fra2/AP-1 in systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to irreversible fibrosis of the skin and the internal organs. The etiology of SSc is complex, its pathophysiology is poorly understood, and clinical therapeutic options are restricted. Thus, research into medications and targets for treating fibrosis is essential and urgent. Fos-related antigen 2 (Fra2) is a transcription factor that is a member of the activator protein-1 family. Fra2 transgenic mice were shown to have spontaneous fibrosis. All-trans retinoic acid (ATRA) is a vitamin A intermediate metabolite and ligand for the retinoic acid receptor (RAR), which possesses anti-inflammatory and anti-proliferative properties. Recent research has demonstrated that ATRA also has an anti-fibrotic effect. However, the exact mechanism is not fully understood. Interestingly, we identified potential binding sites for the transcription factor RARα to the promoter region of the FRA2 gene through JASPAR and PROMO databases. In this study, the pro-fibrotic effect of Fra2 in SSc is confirmed. SSc dermal fibroblasts and bleomycin-induced fibrotic tissues of SSc animals exhibit increased levels of Fra2. Inhibition of Fra2 expression in SSc dermal fibroblasts with Fra2 siRNA markedly decreased collagen I expression. ATRA reduced the expressions of Fra2, collagen I, and α-smooth muscle actin(α-SMA) in SSc dermal fibroblasts and bleomycin-induced fibrotic tissues of SSc mice. In addition, chromatin immunoprecipitation and dual-luciferase assays demonstrated that retinoic acid receptor RARα binds to the FRA2 promoter and modulates its transcriptional activity. ATRA decreases collagen I expression both in vivo and in vitro via the reduction of Fra2 expression. This work establishes the rationale for expanding the use of ATRA in the treatment of SSc and indicates that Fra2 can be used as an anti-fibrotic target.

O-248 Treatment with antifibrotic targets decreases accumulation of pro-fibrotic myofibroblasts and fibrotic collagen in cultured cryopreserved-thawed human cortical tissue

Abstract Study question Could the excess deposition of fibrotic collagen and the presence of myofibroblast be reversed by in vitro culture of human cortical tissue with antifibrotic targets? Summary answer The in vitro treatment of fibrotic ovarian stroma with Metformin, Pirfenidone or Mitoquinone proved effective in decreasing collagen accumulation and the presence of myofibroblasts. What is known already Tissue fibrosis is characterized by an excessive accumulation of extracellular matrix proteins, leading to organ dysfunction. The ovary is the first organ to show fibrosis related to ageing, creating permissive conditions for ovarian cancer development. Due to this, there is an urgent need for ovarian fibrosis treatments. The clinically approved treatment with Metformin has shown efficacy in preventing fibrosis progression from post-menopausal ovaries obtained from diabetic patients, while Pirfenidone is used to treat pulmonary fibrosis. Transmasculine people exposed to prolonged androgen therapy show early signs of ovarian fibrosis, providing a suitable model to study ovarian fibrosis management strategies Study design, size, duration duration Fresh and cryopreserved-thawed human ovarian samples obtained from 9 cisgender women (cOVA) and 9 transgender and gender diverse people (tOVA) were included for histological analysis of collagen content and fiber organization. Cryopreserved-thawed cortical fragments from 6 tOVAs were cultured for 8 days with or without antifibrotic treatments (Metformin, Pirfenidone and Mitoquinone). Cryopreserved-thawed cortical fragments from 9 tOVAs were used for in vitro culture, followed by metabolic assays. Participants/materials, setting, methods Human ovarian samples were obtained from 9 cisgender women (29,1,3±7,9years) scheduled for gonadotoxic therapy undergoing oophorectomy for fertility preservation and from 24 transgender and gender diverse people (22,7±2,4 years) undergoing gender affirming oophorectomy. Collagen content and organization were visualized by second harmonic generation and polarized light imaging. Collagen type 1 and 4, ACTA2+ myofibroblasts and TUNEL+ apoptotic cells were detected and quantified using immunofluorescence. Mitochondrial respiration and glycolysis were measured using the Seahorse XF analyzer. Main results and the role of chance Histological analysis of collagen content and organization confirmed that tOVA from subjects in fertile age presents characteristics of ovarian fibrosis, such as a higher accumulation and anisotropic (linearized) organization of collagen fibers within the stroma, indicating that long exposure to androgen therapy affects the extracellular matrix composition of the ovary. Furthermore, both tOVA and cOVA cortical tissue showed an increase in anisotropic collagen disposition after being cryopreserved-thawed when compared to its fresh counterpart, indicating cryopreservation could favor fibrotic progression. Additionally, standard in vitro culture of cryopreserved-thawed ovarian cortical fragments promotes an accumulation of collagen 1 and 4 as well as an increase in the number of myofibroblasts and apoptotic cells after 8 days of culture. Treatment with antifibrotics targets such as Pirfenidone, Metformin and Mitoquinone proved to be efficient in reducing both collagen accumulation (P &amp;lt; 0.05), pro-fibrotic myofibroblasts (P &amp;lt; 0.05) and TUNEL+ apoptotic cells (P &amp;lt; 0.05). These results support the evidence that ovarian fibrosis can be prevented or reverted in vitro with anti-oxidants and drugs targeting inflammatory response. As such, these treatments should be considered as therapeutic approaches for women of advancing age and metabolic disorders to prevent pro-tumorigenic fibrotic ovarian stroma. Limitations, reasons for caution Small sample size. There is variation in the type and duration of the androgen treatment in transgender and gender diverse people included in the study. Furthermore, the collagen analysis from the cOVA samples was performed on 1cm2 biopsies that might not represent the general collagen distribution in the complete ovary. Wider implications of the findings Medical treatments affecting the ovarian metabolic cell function, such as androgen therapy for transgender people and gender diverse or lab procedures to cryopreserved human ovarian tissue, can trigger fibrosis progression. Nevertheless, human ovarian fibrosis seems reversible and preventable using drugs targeting mitochondrial metabolism and inflammatory response. Trial registration number not applicable

Extracellular Targets to Reduce Excessive Scarring in Response to Tissue Injury.

Excessive scar formation is a hallmark of localized and systemic fibrotic disorders. Despite extensive studies to define valid anti-fibrotic targets and develop effective therapeutics, progressive fibrosis remains a significant medical problem. Regardless of the injury type or location of wounded tissue, excessive production and accumulation of collagen-rich extracellular matrix is the common denominator of all fibrotic disorders. A long-standing dogma was that anti-fibrotic approaches should focus on overall intracellular processes that drive fibrotic scarring. Because of the poor outcomes of these approaches, scientific efforts now focus on regulating the extracellular components of fibrotic tissues. Crucial extracellular players include cellular receptors of matrix components, macromolecules that form the matrix architecture, auxiliary proteins that facilitate the formation of stiff scar tissue, matricellular proteins, and extracellular vesicles that modulate matrix homeostasis. This review summarizes studies targeting the extracellular aspects of fibrotic tissue synthesis, presents the rationale for these studies, and discusses the progress and limitations of current extracellular approaches to limit fibrotic healing.

P008 Fibrostricturing Crohn’s disease is characterised by an imbalance in active eosinophils, Th1, Th2 and regulatory T cells

Abstract Background About one third of patients with Crohn’s disease (CD) develop strictures during their disease course requiring surgical resection. The immune landscape involved in this process is poorly understood. Therefore, we aimed to characterise the fibroblast phenotype, immune cells and their mediators involved in intestinal strictures. Methods We included 25 CD patients with stricturing disease in the terminal ileum (TI) and 10 controls with colorectal cancer (CRC), all undergoing an ileocolonic resection. Transmural samples from the resection specimen of the TI were obtained. Macroscopically, CD tissue was divided into unaffected, fibrostenotic and inflamed regions by an experienced histopathologist. Next, mucosa was separated from deeper layers, after which single cells were isolated and fluorescently stained for flow cytometry. Protein levels were determined via the MesoScale Discovery (MSD) platform in the corresponding samples. Comparisons between CRC controls and CD patients were performed via an unpaired t-test or Mann-Whitney analysis and corrected for multiple testing. Results An increase in active fibroblasts and decrease in inactive fibroblasts in the fibrotic and inflamed mucosa (p=0.0002 and p&amp;lt;0.0001) and deeper layers (p=0.003 and p=0.02) when compared to the CRC controls was observed, confirming ongoing tissue remodelling. An enrichment of active eosinophils was only seen in the fibrotic deeper layers (p=0.02), although an increase in T helper 2 (Th2) cells was observed in both the fibrotic and inflamed deeper layers (p=0.02 and p=0.04). In contrast, T helper 1 (Th1) cells were decreased in both fibrotic and inflamed mucosa (p=0.03 and p=0.02) and deeper layers (p=0.01 for both). Regulatory T cells were significantly enriched in both fibrotic and inflamed mucosa (p&amp;lt;0.0001 and p=0.0005) and deeper layers (p=0.01 and p=0.006) (figure 1). Protein quantification confirmed a significant increase in transforming growth factor-β3 (TGF-β3) in the fibrotic (p=0.007) and inflamed (p=0.0002) layers, but not in the more superficial mucosa. Comparably, IL-1β was increased in the fibrotic (p=0.05) and inflamed (p=0.05) deeper layers. A similar observation was made for basic fibroblast growth factor (bFGF) (p=0.004), although only a trend could be seen in the fibrotic deeper layers (p=0.08) (figure 2). Conclusion The fibrotic and inflamed tissue of CD patients is characterized by increased activated eosinophils, Th2 and regulatory T cells and decreased Th1 cells, as well as many of their mediator cytokines. The current immunological characterisation can help to prioritise potential anti-fibrotic targets for stricturing CD.

An autocrine signaling circuit in hepatic stellate cells underlies advanced fibrosis in nonalcoholic steatohepatitis.

Advanced hepatic fibrosis, driven by the activation of hepatic stellate cells (HSCs), affects millions worldwide and is the strongest predictor of mortality in nonalcoholic steatohepatitis (NASH); however, there are no approved antifibrotic therapies. To identify antifibrotic drug targets, we integrated progressive transcriptomic and morphological responses that accompany HSC activation in advanced disease using single-nucleus RNA sequencing and tissue clearing in a robust murine NASH model. In advanced fibrosis, we found that an autocrine HSC signaling circuit emerged that was composed of 68 receptor-ligand interactions conserved between murine and human NASH. These predicted interactions were supported by the parallel appearance of markedly increased direct stellate cell-cell contacts in murine NASH. As proof of principle, pharmacological inhibition of one such autocrine interaction, neurotrophic receptor tyrosine kinase 3-neurotrophin 3, inhibited human HSC activation in culture and reversed advanced murine NASH fibrosis. In summary, we uncovered a repertoire of antifibrotic drug targets underlying advanced fibrosis in vivo. The findings suggest a therapeutic paradigm in which stage-specific therapies could yield enhanced antifibrotic efficacy in patients with advanced hepatic fibrosis.

Epiregulin is a dendritic cell-derived EGFR ligand that maintains skin and lung fibrosis.

Immune cells are fundamental regulators of extracellular matrix (ECM) production by fibroblasts and have important roles in determining extent of fibrosis in response to inflammation. Although much is known about fibroblast signaling in fibrosis, the molecular signals between immune cells and fibroblasts that drive its persistence are poorly understood. We therefore analyzed skin and lung samples of patients with diffuse cutaneous systemic sclerosis, an autoimmune disease that causes debilitating fibrosis of the skin and internal organs. Here, we define a critical role of epiregulin-EGFR signaling between dendritic cells and fibroblasts to maintain elevated ECM production and accumulation in fibrotic tissue. We found that epiregulin expression marks an inducible state of DC3 dendritic cells triggered by type I interferon and that DC3-derived epiregulin activates EGFR on fibroblasts, driving a positive feedback loop through NOTCH signaling. In mouse models of skin and lung fibrosis, epiregulin was essential for persistence of fibrosis in both tissues, which could be abrogated by epiregulin genetic deficiency or a neutralizing antibody. Therapeutic administration of epiregulin antibody reversed fibrosis in patient skin and lung explants, identifying it as a previously unexplored biologic drug target. Our findings reveal epiregulin as a crucial immune signal that maintains skin and lung fibrosis in multiple diseases and represents a promising antifibrotic target.

HBO1 as an Important Target for the Treatment of CCL4-Induced Liver Fibrosis and Aged-Related Liver Aging and Fibrosis.

The liver is the largest digestive organ in the human body. The increasing incidence of chronic liver fibrosis is one of the major health challenges in the world. Liver fibrosis is a wound-healing response to acute or chronic cellular damage of liver tissue. At present, despite a series of research progress on the pathophysiological mechanism of fibrosis that has been made, there is still a gap in identifying antifibrotic targets and converting them into effective treatments. Therefore, it is extremely important to seek a molecular target that can alleviate or reverse liver fibrosis, which has important scientific and clinical significance. In the current study, to evaluate the therapeutic effect of HBO1 as a molecular target on liver aging and fibrosis, naturally-aged mice and CCL4-induced liver fibrosis mice were used as animal models, and multiple experiments were performed. Experimental results showed that HBO1 knockdown could strongly mitigate the accumulation of hepatic collagen by Masson and Sirius Red staining. Further study showed that HBO1 knockdown reduced the expression of fibrosis-related marker molecules (α-SMA, collagen type I (ColI), and fibronectin). Further work showed that HBO1 knockdown could significantly alleviate HSC activation. On this basis, we analyzed the underlying mechanism by which HBO1 alleviates liver fibrosis. It was found that HBO1 knockdown may modulate liver fibrosis by regulating the processes of EMT, inflammation, and oxidative stress. We further studied the effect of HBO1 knockdown on liver aging and aging-related liver fibrosis, and the results showed that HBO1 knockdown could significantly reduce the level of aging-related liver fibrosis and relieve liver aging. In conclusion, we systematically investigated the potential of HBO1 as a therapeutic target to attenuate liver fibrosis and liver aging. The current study found a crucial target for liver fibrosis and liver-aging therapy, which has laid a solid foundation for the liver fibrosis-related research.

Epigenetic control of LncRNA NEAT1 enables cardiac fibroblast pyroptosis and cardiac fibrosis

Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after inflammatory injury, leading to cardiac fibrosis and diastolic dysfunction. Recent studies described the role of epigenetics in cardiac fibrosis. Nevertheless, detailed reports on epigenetics regulating CFs pyroptosis and describing their implication in cardiac fibrosis are still unclear. Here, we found that DNMT3A reduces the expression of lncRNA Neat1 and promotes the NLRP3 axis leading to CFs pyroptosis, using cultured cells, animal models, and clinical samples to shed light on the underlying mechanism. We report that pyroptosis-related genes are increased explicitly in cardiac fibrosis tissue and LPS-treated CFs, while lncRNA Neat1 decreased. Mechanistically, we show that loss of DNMT3A or overexpression of lncRNA Neat1 in CFs after LPS treatment significantly enhances CFs pyroptosis and the production of pyroptosis-related markers in vitro. It has been demonstrated that DNMT3A can decrease lncRNA Neat1, promoting NLRP3 axis activation in CFs treated with LPS. In sum, this study is the first to identify that DNMT3A methylation decreases the expression of lncRNA Neat1 and promotes CFs pyroptosis and cardiac fibrosis, suggesting that DNMT3A and NEAT1 may function as an anti-fibrotic therapy target in cardiac fibrosis.