Preventing fibrosis in IBD: update on immune pathways and clinical strategies.

INTRODUCTION: Intestinal fibrosis is a common and serious complication of inflammatory bowel diseases (IBD) driving stricture formation in Crohn's disease patients and leading to submucosal damage in ulcerative colitis. Recent studies provided novel insights into the role of immune and nonimmune components in the pathogenesis of intestinal fibrosis. Those new findings may accelerate the development of anti-fibrotic treatment in IBD patients. AREAS COVERED: This review is designed to cover the recent progress in mechanistic research and therapeutic developments on intestinal fibrosis in IBD patients, including new cell clusters, cytokines, proteins, microbiota, creeping fat, and anti-fibrotic therapies. EXPERT OPINION: Due to the previously existing major obstacle of missing consensus on stricture definitions and the absence of clinical trial endpoints, testing of drugs with an anti-fibrotic mechanism is just starting in stricturing Crohn's disease (CD). A biomarker to stratify CD patients at diagnosis without any complications into at-risk populations for future strictures would be highly desirable. Further investigations are needed to identify novel mechanisms of fibrogenesis in the intestine that are targetable and ideally gut specific.

Hitting the Target! Challenges and Opportunities for TGF-ß Inhibition for the Treatment of Cardiac fibrosis.

Developing effective anti-fibrotic therapies for heart diseases holds the potential to address unmet needs in several cardiac conditions, including heart failure with preserved ejection fraction, hypertrophic cardiomyopathy, and cardiotoxicity induced by cancer therapy. The inhibition of the primary fibrotic regulator, transforming growth factor (TGF) ß, represents an efficient strategy for mitigating fibrosis in preclinical models. However, translating these findings into clinical benefits faces challenges due to potential adverse effects stemming from TGF-ß's physiological actions in inflammation and tissue homeostasis. Various strategies exist for inhibiting TGF-ß, each associated with a distinct risk of adverse effects. Targeting TGF-ß directly or through its signaling pathway proves efficient in reducing fibrosis. However, direct TGF-ß blockade may lead to uncontrolled inflammation, especially following myocardial infarction, while interference with the signaling pathway may compromise structural integrity, resulting in issues like insufficient wound healing or ventricular dilatation. Influencing TGF-ß activity through interacting signaling pathways, for instance by inhibitors of the renin-angiotensin-aldosterone-system, is insufficiently potent in reducing fibrosis. Targeting activators of latent TGF-ß, including ADAMTS enzymes, thrombospondin, and integrins, emerges as a potentially safer strategy to reduce TGF-ß-induced fibrosis but it requires the identification of appropriate targets. Encouragement is drawn from promising agents developed for fibrosis in other organs, fueling hope for similar breakthroughs in treating cardiac fibrosis. Such advances depend on overcoming obstacles for the implementation of anti-fibrotic strategies in patients with heart disease, including fibrosis quantification. In this review, insights garnered from interventional and mechanistic studies, obtained through a non-systemic search spanning preclinical and clinical evidence, are summarized to pinpoint the most promising targets for further exploration and development.

HSPB5 Inhibition by NCI-41356 Reduces Experimental Lung Fibrosis by Blocking TGF-ß1 Signaling.

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-ß gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-ß1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-ß1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-ß1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-ß1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-ß1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

Progressive stages of dysmetabolism are associated with impaired biological features of human cardiac stromal cells mediated by the oxidative state and autophagy.

Cardiac stromal cells (CSCs) are the main players in fibrosis. Dysmetabolic conditions (metabolic syndrome-MetS, and type 2 diabetes mellitus-DM2) are strong pathogenetic contributors to cardiac fibrosis. Moreover, modulation of the oxidative state (OxSt) and autophagy is a fundamental function affecting the fibrotic commitment of CSCs, that are adversely modulated in MetS/DM2. We aimed to characterize CSCs from dysmetabolic patients, and to obtain a beneficial phenotypic setback from such fibrotic commitment by modulation of OxSt and autophagy. CSCs were isolated from 38 patients, stratified as MetS, DM2, or controls. Pharmacological modulation of OxSt and autophagy was obtained by treatment with trehalose and NOX4/NOX5 inhibitors (TREiNOX). Flow-cytometry and real-time quantitative polymerase chain reaction (RT-qPCR) analyses showed significantly increased expression of myofibroblasts markers in MetS-CSCs at baseline (GATA4, ACTA2, THY1/CD90) and after starvation (COL1A1, COL3A1). MetS- and DM2-CSCs displayed a paracrine profile distinct from control cells, as evidenced by screening of 30 secreted cytokines, with a significant reduction in vascular endothelial growth factor (VEGF) and endoglin confirmed by enzyme-linked immunoassay (ELISA). DM2-CSCs showed significantly reduced support for endothelial cells in angiogenic assays, and significantly increased H2 O2 release and NOX4/5 expression levels. Autophagy impairment after starvation (reduced ATG7 and LC3-II proteins) was also detectable in DM2-CSCs. TREiNOX treatment significantly reduced ACTA2, COL1A1, COL3A1, and NOX4 expression in both DM2- and MetS-CSCs, as well as GATA4 and THY1/CD90 in DM2, all versus control cells. Moreover, TREiNOX significantly increased VEGF release by DM2-CSCs, and VEGF and endoglin release by both MetS- and DM2-CSCs, also recovering the angiogenic support to endothelial cells by DM2-CSCs. In conclusion, DM2 and MetS worsen microenvironmental conditioning by CSCs. Appropriate modulation of autophagy and OxSt in human CSCs appears to restore these features, mostly in DM2-CSCs, suggesting a novel strategy against cardiac fibrosis in dysmetabolic patients. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Animal models and drug candidates for use in glaucoma filtration surgery: A systematic review.

Glaucoma, a degenerative disease of the optic nerve, is the leading cause of irreversible blindness worldwide. Currently, there is no curative treatment. The only proven treatment is lowering intraocular pressure (IOP), the most important risk factor. Glaucoma filtration surgery (GFS) can effectively lower IOP. However, approximately 10% of all surgeries fail yearly due to excessive wound healing, leading to fibrosis. GFS animal models are commonly used for the development of novel treatment modalities. The aim of the present review was to provide an overview of available animal models and anti-fibrotic drug candidates. MEDLINE and Embase were systematically searched. Manuscripts until September 1st, 2021 were included. Studies that used animal models of GFS were included in this review. Additionally, the snowball method was used to identify other publications which had not been identified through the systematic search. Two hundred articles were included in this manuscript. Small rodents (e.g. mice and rats) are often used to study the fibrotic response after GFS and to test drug candidates. Due to their larger eyes, rabbits are better suited to develop medical devices. Novel drugs aim to inhibit specific pathways, e.g. through the use of modulators, monoclonal antibodies, aqueous suppressants or gene therapy. Although most newly studied drugs offer a higher safety profile compared to antimetabolites, their efficacy is in most cases lower when compared to MMC. Current literature on animal models and potential drug candidates for GFS were summarized in this review. Future research should focus on refining current animal models (for example through the induction of glaucoma prior to undertaking GFS) and standardizing animal research to ensure a higher reproducibility and reliability across different research groups. Lastly, novel therapies need to be further optimized, e.g. by conducting more research on the dosage, administration route, application frequency, the option of creating combination therapies, or the development of drug delivery systems for sustained release of anti-fibrotic medication.

Virus infection induced pulmonary fibrosis.

Pulmonary fibrosis is the end stage of a broad range of heterogeneous interstitial lung diseases and more than 200 factors contribute to it. In recent years, the relationship between virus infection and pulmonary fibrosis is getting more and more attention, especially after the outbreak of SARS-CoV-2 in 2019, however, the mechanisms underlying the virus-induced pulmonary fibrosis are not fully understood. Here, we review the relationship between pulmonary fibrosis and several viruses such as Human T-cell leukemia virus (HTLV), Human immunodeficiency virus (HIV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Murine γ-herpesvirus 68 (MHV-68), Influenza virus, Avian influenza virus, Middle East Respiratory Syndrome (MERS)-CoV, Severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 as well as the mechanisms underlying the virus infection induced pulmonary fibrosis. This may shed new light on the potential targets for anti-fibrotic therapy to treat pulmonary fibrosis induced by viruses including SARS-CoV-2.

Idiopathic pulmonary fibrosis-A challenging disease with new horizons and changing therapeutic landscape.

OBJECTIVES: To discuss the pathophysiological hypotheses of IPF with a view to summarise the data on pharmacological aspects of treatment of this fibrotic Interstitial Lung Disease. Furthermore, the adverse effects are briefly discussed for the currently available and licenced anti-fibrotic agents. DATA SOURCE: The data were obtained from the Randomised Controlled Trials and scientific studies published in English literature. The manuscript is kept brief to provide an overview of pathophysiological and pharmacological interplay involved in IPF and it was aimed not to be exhaustive so an update is provided on the aspect of pharmacotherapeutics for physicians involved in managing patients with IPF. RESULTS: Two pharmacological agents Pirfenidone and Nintedanib are discussed with the evidence backing up for the rationale of these drugs to slow the disease progression and potentially improve mortality in this disease with a dismal prognosis. The drugs are associated with adverse events and a careful consideration to balance the efficacy with quality of life of individual patient should be considered before commencing these medications. We are seeing real-world data on the value of these anti-fibrotic agents and there is further evidence of them to be efficacious in severe disease and even in the cohort with significant progression over 6-12 months period. CONCLUSION: The landscape of IPF management has seen a paradigm shift in the last decade form anti-inflammatory to anti-fibrotic approach and with evidence of slowing disease progression. We are likely to improve IPF management in the near future with multi-modality and personalised therapy.

Serum biomarker CA 15-3 as predictor of response to antifibrotic treatment and survival in idiopathic pulmonary fibrosis.

Aim: Cancer antigen 15-3 (CA 15-3) is a baseline biomarker in idiopathic pulmonary fibrosis (IPF), but its value during follow-up is unknown. Materials and methods: Associations between serum CA 15-3 and pulmonary function tests during 1-year follow-up were evaluated by a mixed model in 132 IPF treated with pirfenidone or nintedanib. Results: Increased baseline (median: 56 kU/l) and follow-up CA 15-3 levels were inversely associated with forced vital capacity and diffusing capacity of the lung for carbon monoxide (estimates respectively: -5.21 and -4.69; p < 0.001). Baseline and 6-month CA 15-3 above 58.5 (hazard ratio: 1.67; p = 0.031) and 50.5 kU/l (hazard ratio: 2.99; p < 0.001), respectively, showed impaired survival compared with lower levels. Conclusion: CA 15-3 is associated with pulmonary function test during follow-up in IPF on antifibrotic treatment. Higher (follow-up) values are related with poor survival. Therefore, CA 15-3 is a promising follow-up biomarker in IPF.

Treatment of idiopathic pulmonary fibrosis with Nintedanib: an update.

Introduction: Idiopathic pulmonary fibrosis (IPF) is an incurable, progressive and debilitating disease. Nintedanib is one of two anti-fibrotic therapies available for the treatment of IPF and has been approved since 2014. Together with pirfenidone and antacid medications it has received a conditional recommendation for the treatment for IPF by international clinical practice guidelines.Areas covered: The authors review the mechanisms of action, pharmacological profile and update scientific data and our opinions on efficacy, safety profile and tolerability of nintedanib.Expert opinion: Nintedanib significantly slows disease progression in IPF patients with tolerable and manageable side effects. Its potential future role in the treatment of progressive fibrosing interstitial lung diseases other than IPF is challenging.

Therapeutic effects of serum extracellular vesicles in liver fibrosis.

The lack of approved therapies for hepatic fibrosis seriously limits medical management of patients with chronic liver disease. Since extracellular vesicles (EVs) function as conduits for intercellular molecular transfer, we investigated if EVs from healthy individuals have anti-fibrotic properties. Hepatic fibrogenesis or fibrosis in carbon tetrachloride (CCl4)- or thioacetic acid-induced liver injury models in male or female mice were suppressed by serum EVs from normal mice (EVN) but not from fibrotic mice (EVF). CCl4-treated mice undergoing EVN therapy also exhibited reduced levels of hepatocyte death, inflammatory infiltration, circulating AST/ALT levels and hepatic or circulating pro-inflammatory cytokines. Hepatic histology, liver function tests or circulating proinflammatory cytokine levels were unaltered in control mice receiving EVN. As determined using PKH26-labelled EVN, principal target cells included hepatic stellate cells (HSC; a normally quiescent fibroblastic cell that undergoes injury-induced activation and produces fibrosis during chronic injury) or hepatocytes which showed increased EVN binding after, respectively, activation or exposure to CCl4. In vitro, EVN decreased proliferation and fibrosis-associated molecule expression in activated HSC, while reversing the inhibitory effects of CCl4 or ethanol on hepatocyte proliferation. In mice, microRNA-34c, -151-3p, -483-5p, -532-5p and -687 were more highly expressed in EVN than EVF and mimics of these microRNAs (miRs) individually suppressed fibrogenic gene expression in activated HSC. A role for these miRs in contributing to EVN actions was shown by the ability of their corresponding antagomirs to individually and/or collectively block the therapeutic effects of EVN on activated HSC or injured hepatocytes. Similarly, the activated phenotype of human LX-2 HSC was attenuated by serum EVs from healthy human subjects and contained higher miR-34c, -151-3p, -483-5p or -532-5p than EVs from hepatic fibrosis patients. In conclusion, serum EVs from normal healthy individuals are inherently anti-fibrogenic and anti-fibrotic, and contain microRNAs that have therapeutic actions in activated HSC or injured hepatocytes. Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; CCl4: carbon tetrachloride; CCN2: connective tissue growth factor; E: eosin; EGFP: enhanced green fluorescent protein; EVs: extracellular vesicles; EVF: serum EVs from mice with experimental hepatic fibrosis; EVN: serum EVs from normal mice; H: hematoxylin; HSC: hepatic stellate cell; IHC: immunohistochemistry; IL: interleukin; MCP-1: monocyte chemotactic protein-1; miR: microRNA; mRNA: messenger RNA; NTA: nanoparticle tracking analysis; PCNA: proliferating cell nuclear antigen; qRT-PCR: quantitative real-time polymerase chain reaction; SDS-PAGE: sodium dodecyl sulphate - polyacrylamide gel electrophoresis; αSMA: alpha smooth muscle actin; TAA: thioacetic acid; TG: transgenic; TGF-ß: transforming growth factor beta; TEM: transmission electron microscopy; TNFα: tumour necrosis factor alpha.

Outcome of lung transplantation in idiopathic pulmonary fibrosis with previous anti-fibrotic therapy.

BACKGROUND: Anti-fibrotic drugs may interfere with wound-healing after major surgery, theoretically preventing sufficient bronchial anastomosis formation after lung transplantation (LTx). The aim of this study was to assess the impact of previous treatment with pirfenidone and nintedanib on outcomes after LTx in patients with idiopathic pulmonary fibrosis (IPF). METHODS: All patients with IPF undergoing LTx at the University of Munich between January 2012 and November 2016 were retrospectively screened for previous use of anti-fibrotics. Post-transplant outcome and survival of patients with and without anti-fibrotic treatment were analyzed. RESULTS: A total of 62 patients with IPF were transplanted (lung allocation score [mean ± SD] 53.1 ± 16.1). Of these, 23 (37.1%) received pirfenidone and 7 (11.3%) received nintedanib before LTx; the remaining 32 (51.6%) did not receive any anti-fibrotic drug (control group). Patients receiving anti-fibrotics were significantly older (p = 0.004) and their carbon monoxide diffusion capacity was significantly higher (p = 0.008) than in controls. Previous anti-fibrotic treatment did not increase blood product utilization, wound-healing or anastomotic complications after LTx. Post-transplant surgical revisions due to bleeding and/or impaired wound-healing were necessary in 18 (29.0%) patients (pirfenidone 30.4%, nintedanib 14.3%, control 31.3%; p = 0.66). Anastomosis insufficiency occurred in 2 (3.2%) patients, both in the control group. No patient died within the first 30 days post-LTx, and no significant differences regarding survival were seen during the follow-up (12-month survival: pirfenidone 77.0%, nintedanib 100%, control 90.6%; p = 0.29). CONCLUSION: Our data show that previous use of anti-fibrotic therapy does not increase surgical complications or post-operative mortality after LTx.

24-nor-ursodeoxycholic acid ameliorates inflammatory response and liver fibrosis in a murine model of hepatic schistosomiasis.

BACKGROUND & AIMS: Intrahepatic granuloma formation and fibrosis characterize the pathological features of Schistosoma mansoni infection. Based on previously observed substantial anti-fibrotic effects of 24-nor-ursodeoxycholic acid (norUDCA) in Abcb4/Mdr2(-/-) mice with cholestatic liver injury and biliary fibrosis, we hypothesized that norUDCA improves inflammation-driven liver fibrosis in S. mansoni infection. METHODS: Adult NMRI mice were infected with 50 S. mansoni cercariae and after 12 weeks received either norUDCA- or ursodeoxycholic acid (UDCA)-enriched diet (0.5% wt/wt) for 4 weeks. Bile acid effects on liver histology, serum biochemistry, key regulatory cytokines, hepatic hydroxyproline content as well as granuloma formation were compared to naive mice and infected controls. In addition, effects of norUDCA on primary T-cell activation/proliferation and maturation of the antigen-presenting-cells (dendritic cells, macrophages) were determined in vitro. RESULTS: UDCA as well as norUDCA attenuated the inflammatory response in livers of S. mansoni infected mice, but exclusively norUDCA changed cellular composition and reduced size of hepatic granulomas as well as TH2-mediated hepatic fibrosis in vivo. Moreover, norUDCA affected surface expression level of major histocompatibility complex (MHC) class II of macrophages and dendritic cells as well as activation/proliferation of T-lymphocytes in vitro, whereas UDCA had no effect. CONCLUSIONS: This study demonstrates pronounced anti-inflammatory and anti-fibrotic effects of norUDCA compared to UDCA in S. mansoni induced liver injury, and indicates that norUDCA directly represses antigen presentation of antigen presenting cells and subsequent T-cell activation in vitro. Therefore, norUDCA represents a promising drug for the treatment of this important cause of liver fibrosis.

The crucial role of cholangiocytes in cholangiopathies.

Cholangiopathies are diseases involving the intrahepatic biliary tree. They appear to involve, chronic inflammation of the bile ducts, which can lead to the development of bile duct cholestasis, proliferation/ductopenia, biliary fibrosis, and malignant transformation. Sustained stimulatory insults to biliary epithelial cells can induce a ductular reaction, which has a key role in the initiation and progression of cholangiopathies. The epithelial-mesenchymal interaction between reactive cholangiocytes and mesenchymal cells with the inflammatory infiltrates plays a major role in this pathogenesis. Cytokines, chemokines, growth factors and morphogens mediate these interactions in an autocrine or paracrine manner. The main hepatic myofibroblasts (MFs) in cholangiopathies originate from portal fibroblasts. Hepatic stellate cells and fibrocytes also transform into MFs. Whether cholangiocytes or hepatocytes are a source of MFs via the epithelial-mesenchymal transition (EMT) remains a matter of controversy. Although there have been numerous indirect findings supporting the theory of a cholangiocyte EMT in human tissues, recent studies using lineage tracing methods have demonstrated strong evidence against the EMT. Understanding the pathogenic mechanisms involved in cholangiopathies can allow for better-targeted anti-fibrotic therapies in animal models. Before anti-fibrotic therapies can translate into clinical trials, improved monitoring of the fibrotic progression of cholangiopathies and an accurate assessment regarding the effectiveness of the proposed treatments must be achieved.

CCN3/CCN2 regulation and the fibrosis of diabetic renal disease.

Prior work in the CCN field, including our own, suggested to us that there might be co-regulatory activity and function as part of the actions of this family of cysteine rich cytokines. CCN2 is now regarded as a major pro-fibrotic molecule acting both down-stream and independent of TGF-beta1, and appears causal in the disease afflicting multiple organs. Since diabetic renal fibrosis is a common complication of diabetes, and a major cause of end stage renal disease (ESRD), we examined the possibility that CCN3 (NOV), might act as an endogenous negative regulator of CCN2 with the capacity to limit the overproduction of extracellular matrix (ECM), and thus prevent, or ameliorate fibrosis. We demonstrate, using an in vitro model of diabetic renal fibrosis, that both exogenous treatment with CCN3 and transfection with the over-expression of the CCN3 gene in mesangial cells markedly down-regulates CCN2 activity and blocks ECM over-accumulation stimulated by TGF-beta1. Conversely, TGF-beta1 treatment reduces endogenous CCN3 expression and increases CCN2 activity and matrix accumulation, indicating an important, novel yin/yang effect. Using the db/db mouse model of diabetic nephropathy, we confirm the expression of CCN3 in the kidney, with temporal localization that supports these in vitro findings. In summary, the results corroborate our hypothesis that one function of CCN3 is to regulate CCN2 activity and at the concentrations and conditions used down-regulates the effects of TGF-beta1, acting to limit ECM turnover and fibrosis in vivo. The findings suggest opportunities for novel endogenous-based therapy either by the administration, or the upregulation of CCN3.