Fibrosis In Experimental Model Research Articles

Experimental models of fibrosis

Fibrosis, as a pathological process, is characterized by excessive accumulation of extracellular matrix and can affect various organs and tissues, including the lungs, liver, heart and kidneys, leading to serious morbidity and poor quality of life. The main mechanisms of fibrosis are disturbances in cellular signaling pathways, their regulation, interaction, including disturbances in the exchange of cellular regulatory signals, impaired mechanisms of cell adhesion, and changes in the extracellular matrix. All this makes the search for new agents with antifibrotic activity to be urgent. The review examines the basic mechanisms of the development of fibrosis with an emphasis on experimental models, as well as the potential and limitations of experimental models of fibrosis in the context of further search and study of new drugs with antifibrotic activity.

Mutual Amplification of GLI2/Hedgehog and Transcription Factor JUN/AP-1 Signaling in Fibroblasts in Systemic Sclerosis: Potential Implications for Combined Therapies.

Deregulation of the cJUN/AP-1 and hedgehog/GLI2 signaling pathways has been implicated in fibroblast activation in systemic sclerosis (SSc). However, the consequences of their concomitant up-regulation are unknown. Here, we tested the hypothesis that mutual amplification of both pathways might drive persistent fibroblast activation. Cultured fibroblasts and skin sections of patients with diffuse SSc and healthy volunteers were analyzed. cJUN/AP-1 signaling and hedgehog/GLI2 signaling were inhibited using knockdown and pharmacologic approaches. Hedgehog signaling was activated in mice by fibroblast-specific overexpression of constitutively active Smoothened. cJUN and GLI2 are concomitantly up-regulated and colocalize in fibroblasts of patients with SSc compared to healthy controls. Activation of hedgehog/GLI2 signaling induces the expression of cJUN in vitro and in vivo, whereas inactivation of GLI2 inhibits cJUN expression. Likewise, inactivation of cJUN impairs the expression of GLI2. This mutual regulation occurs at the level of transcription with binding of cJUN and GLI2 to specific binding motifs. Interference with this mutual amplification of cJUN signaling and GLI2 signaling inhibits fibroblast activation and collagen release: Inhibition of cJUN/AP-1 signaling ameliorates hedgehog-induced fibroblast activation and skin fibrosis in SmoACT mice with a reduction of skin thickness of 103% (P = 0.0043) in the treatment group compared to the fibrotic control group. Moreover, combined pharmacologic inhibition of cJUN/AP-1 and hedgehog/GLI2 exerts additive antifibrotic effects in a model of TGFβ-driven experimental fibrosis (TBRACT mice). The transcription factors cJUN and GLI2 reinforce each other's activity to promote fibroblast activation in SSc. Interruption of this crosstalk by combined inhibition of both pathways exerts additive antifibrotic effects at well-tolerated doses.

Increased NPM1 inhibit ferroptosis and aggravate renal fibrosis via Nrf2 pathway in chronic kidney disease

Recent findings underscore the significance of ferroptosis, an innovative iron-dependent mode of cell death, in the etiology and progression of chronic kidney disease (CKD). Nucleophosmin 1 (NPM1), a nucleolar protein, contributes to fibrogenesis and modulates cellular functions and mortality. Initial investigations utilized bioinformatics techniques to pinpoint genes with altered expression in CKD and to forecast the potential links between NPM1, ferroptosis, and renal fibrosis. Increased NPM1 expression was verified in the renal tissues of CKD patients. Experimental models of renal fibrosis in both animals and cells were then used for further study. The suppression of NPM1 led to an augmentation in iron metabolism and lipid peroxidation processes integral to ferroptosis, contributing to the mitigation of renal fibrosis. In contrast, an elevation in NPM1 expression had the opposite effect. This modulation may be interconnected with the nuclear factor erythroid 2–related factor 2 pathway. Moreover, the application of the ferroptosis inhibitor, Fer-1, not only obstructed ferroptosis but also diminished NPM1 expression, which, in turn, contributed to the alleviation of renal fibrosis. Thus, our findings suggest that in CKD the NPM1 level increased and led to decreased ferroptosis and aggravated renal fibrosis via an Nrf2 pathway. Ferroptosis inhibitor can alleviate renal fibrosis.

Updates on the controversial roles of regulatory lymphoid cells in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and severe form of pulmonary fibrosis, characterized by scar formation in the lung interstitium. Transforming growth factor beta (TGF-β) is known as a key mediator in the fibrotic process, acting on fibroblasts and mediating their proliferation and differentiation into myofibroblasts. Although the immune system is not considered responsible for the initiation of IPF, markers of tolerogenic immunity define the pro-fibrotic microenvironment in the lungs. In homeostatic conditions, regulatory T cells (Tregs) constitute the main lymphoid population responsible for maintaining peripheral tolerance. Similar to Tregs, regulatory B cells (Bregs) represent a recently described subset of B lymphocytes with immunosuppressive functions. In the context of IPF, numerous studies have suggested a role for Tregs in enhancing fibrosis, mainly via the secretion of TGF-β. In humans, most studies show increased percentages of Tregs associated with the severity of IPF, although their exact role remains unclear. In mice, the most commonly used model involves triggering acute lung inflammation with bleomycin, leading to a subsequent fibrotic process. Consequently, data are still conflicting, as Tregs may play a protective role during the inflammatory phase and a deleterious role during the fibrotic phase. Bregs have been less studied in the context of IPF, but their role appears to be protective in experimental models of lung fibrosis. This review presents the latest updates on studies exploring the implication of regulatory lymphoid cells in IPF and compares the different approaches to better understand the origins of conflicting findings.

Insights on the Tunisian Prickly Pear Molasses as a Potential Antifibrotic and Antioxidant Candidate against Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial disease leading to pulmonary damage and respiratory failure. We aimed to investigate the effect of prickly pear molasses (PPM) on an experimental model of pulmonary fibrosis induced by bleomycin (BLM) in Wistar rat. Animals were divided into 5 groups: the control group (G1), the BLM group (G2) and three groups (G3, G4, G5) receiving a single intra-tracheal injection of BLM (4 mg/kg) and PPM (at 2, 4.5 and10 %) that was introduced into the diet one week before BLM injection and continued for 3 weeks. Our phytochemical results revealed significant polyphenol and flavonoid content. LCMS analysis revealed the presence of Sinapinic acid, t-ferulic acid, t-cinnamic acid, Caffeic acid, gallic acid and vallinic acid among others. Our histological study revealed significant decrease in collagen deposition in the groups of rats treated with 4.5 % and 10 % molasses compared to BLM group. Oxidative stress in pulmonary tissues was investigated using catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) assays. Treatment with PPM normalized the disturbance in the level of these oxidative markers in G3,G4, G5 compared to G2. In conclusion, PPM exhibit antifibrotic and antioxidant activities in BLM model of lung fibrosis.

Cellular and Molecular Genetic Mechanisms of Lung Fibrosis Development and the Role of Vitamin D: A Review.

Idiopathic pulmonary fibrosis remains a relevant problem of the healthcare system with an unfavorable prognosis for patients due to progressive fibrous remodeling of the pulmonary parenchyma. Starting with the damage of the epithelial lining of alveoli, pulmonary fibrosis is implemented through a cascade of complex mechanisms, the crucial of which is the TGF-β/SMAD-mediated pathway, involving various cell populations. Considering that a number of the available drugs (pirfenidone and nintedanib) have only limited effectiveness in slowing the progression of fibrosis, the search and justification of new approaches aimed at regulating the immune response, cellular aging processes, programmed cell death, and transdifferentiation of cell populations remains relevant. This literature review presents the key modern concepts concerning molecular genetics and cellular mechanisms of lung fibrosis development, based mainly on in vitro and in vivo studies in experimental models of bleomycin-induced pulmonary fibrosis, as well as the latest data on metabolic features, potential targets, and effects of vitamin D and its metabolites.

Experimental mouse model of pulmonary fibrosis induced by nebulized LPS administration

Lipopolysaccharide (LPS)-induced lung injury is the most commonly used mouse model of acute lung inflammation that simulates the development of respiratory distress syndrome in humans. The effects of acute LPS-induced airway inflammation are well studied and associated with the neutrophil accumulation in bronchoalveolar lavage fluid (BALF), local and systemic production of proinflammatory cytokines and narrowing of the airways. Recent studies demonstrated the presence of pulmonary fibrosis characterized by increased fibroblast proliferation and excess extracellular matrix deposition in late phase of acute lung inflammation caused by LPS exposure. This work describes an experimental model of acute lung injury induced by a single aerosol injection of LPS as a reproducible in vivo model of pulmonary fibrosis. To induce lung injury, C57BL/6 mice were placed in a chamber and exposed to an aerosol containing 10 mg of LPS using an Aeroneb Lab Nebulizer delivery system. We found that 5 weeks after a single nebulized LPS administration, mice have increased production of IL-6 in BALF. Although the frequency of neutrophils was not altered, there was a decrease in the percentage of alveolar macrophages at 5 weeks after LPS exposure, indicating continued lung inflammation. Several weeks after aerosolized LPS challenge, IL-10 production in BALF was increased, as well as expression of Tgfb1, Col1a1, Il13 and Acta2, and collagen deposition in lung tissue compared to mice with acute lung inflammation. Thus, the single nebulized LPS administration represents a relevant, reproducible and physiologic model in mice allowing to investigate the mechanisms of pulmonary fibrosis development and help in the search for new therapeutic agents and approaches.

Protective Effects of Tunisian Orange Co-Product Extract and Oleuropein-Hesperidin Combination on Bleomycin-Induced Pulmonary Fibrosis in Rats.

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia that leads to acute lung damage, deterioration of lung function, and increased mortality risk. In this study, we investigated the effects of the orange coproduct extract (OCE) and the combination of pure hesperidin and oleuropein (HO) on an experimental model of pulmonary fibrosis induced by bleomycin (BLM) in Wistar rats. Rats were divided into six groups: the control group (G1), the BLM group (G2), three groups (G3, G4, G5) receiving a single dose of BLM combined with OCE extract at 100, 200, and 300 mg/kg, and group 6 (G6) receiving a single dose of BLM combined with HO: both pure major phenolic compounds of OCE (hesperidin at 50 mg/kg) and olive leaves (oleuropein at 2.5 mg/kg). Oxidative stress in lung tissues was investigated using catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX) assays and the measurement of malondialdehyde (MDA) and lactate dehydrogenase (LDH) levels. Treatment with OCE and HO normalized the disturbance in oxidative markers' levels and showed a significant reduction in fibrosis score with no renal or hepatic toxic effects. In conclusion, OCE and HO exhibit antifibrotic effects on a rat model of pulmonary fibrosis.

Vinpocetine alleviated alveolar epithelial cells injury in experimental pulmonary fibrosis by targeting PPAR-γ/NLRP3/NF-κB and TGF-β1/Smad2/3 pathways

This study aimed to investigate the potential anti-fibrotic activity of vinpocetine in an experimental model of pulmonary fibrosis by bleomycin and in the MRC-5 cell line. Pulmonary fibrosis was induced in BALB/c mice by oropharyngeal aspiration of a single dose of bleomycin (5 mg/kg). The remaining induced animals received a daily dose of pirfenidone (as a standard anti-fibrotic drug) (300 mg/kg/PO) and vinpocetine (20 mg/kg/PO) on day 7 of the induction till the end of the experiment (day 21). The results of the experiment revealed that vinpocetine managed to alleviate the fibrotic endpoints by statistically improving (P ≤ 0.05) the weight index, histopathological score, reduced expression of fibrotic-related proteins in immune-stained lung sections, as well as fibrotic markers measured in serum samples. It also alleviated tissue levels of oxidative stress and inflammatory and pro-fibrotic mediators significantly elevated in bleomycin-only induced animals (P ≤ 0.05). Vinpocetine managed to express a remarkable attenuating effect in pulmonary fibrosis both in vivo and in vitro either directly by interfering with the classical TGF-β1/Smad2/3 signaling pathway or indirectly by upregulating the expression of Nrf2 enhancing the antioxidant system, activating PPAR-γ and downregulating the NLRP3/NF-κB pathway making it a candidate for further clinical investigation in cases of pulmonary fibrosis.

Ciplukan (Physalis angulata Linn) Bioactivity against Bleomycin-Induced Pulmonary Fibrosis in Mice by reducing Subpleural Fibrosis, KL-6 Level and Anti-Inflammatory properties

Pulmonary fibrosis is a fatal lung disease with unknown pathogenesis and limited treatment options. Herbal medicine began to be developed as an antifibrosis drug for this disease. Ciplukan plant (Physalis angulata Linn.), is a wild plant that has been widely used for generations as traditional Indonesian medicine for various diseases; but has never been studied as an antifibrosis. This study aimed to determine Ciplukan herb ethanol extract (CPL) bioactivity as antifibrosis in pulmonary fibrosis disorders in experimental mice model induced by bleomycin. A total of 35 male mice and 35 female mice of the ddy strain was divided into 7 groups respectively with 1 normal control group and 6 experimental animal models of pulmonary fibrosis induced by bleomycin groups. For the pulmonary fibrosis model, bleomycin (BLM) was injected subcutaneously 8 times with a frequency of twice a week for 4 weeks. Furthermore, the mice were given CPL orally starting at week 6 of treatment with 2 different doses, 1.95mg (CPL-1) and 3.9mg (CPL) every day for 4 weeks. Pulmonary fibrosis histopathology was analyzed using HE and MT staining methods. Serum IL-6, KL-6, and TGF-β1 levels determination was carried out using the ELISA method. The administration of CPL significantly reduced the fibrosis score from 2.80±1.095 to 1.67±0.577µm (p=0.026). The CPL also showed anti-inflammatory activity by reducing IL-6 levels from 1916.20±594.27 to 16.81±17.07pg/mL (p=0.003); TGF-β1 levels from 51.25±2.25 to 22.48±0.93ng/mL (p=0.021); and KL-6 levels from 28.09±2.25 to 13.99±0.93ng/mL (p=0.000). CPL was proven to have pulmonary antifibrotic activity in experimental mice model. The pulmonary antifibrotic effect was evidenced by a decrease in pulmonary fibrosis scores also a decrease in KL-6 levels, IL-6 levels, and TGF-β1. The administration of CPL-1 and CPL-2 can provide recovery of pulmonary fibrosis induced by bleomycin.

Pathogenesis and management of renal fibrosis induced by unilateral ureteral obstruction.

Regardless of the underlying etiology, renal fibrosis is the final histological outcome of progressive kidney disease. Unilateral ureteral obstruction (UUO) is an ideal and reproducible experimental rodent model of renal fibrosis, which is characterized by tubulointerstitial inflammatory responses, accumulation of extracellular matrix, tubular dilatation and atrophy, and fibrosis. The magnitude of UUO-induced renal fibrosis is experimentally manipulated by the species chosen, animal age, and the severity and duration of the obstruction, while relief of the obstruction allows the animal to recover from fibrosis. The pathogenesis of renal fibrosis is complex and multifactorial and is orchestrated by activation of renin-angiotensin system (RAS), oxidative stress, inflammatory response, transforming growth factor beta 1-Smad pathway, activated myofibroblasts, cell death (apoptosis, autophagy, ferroptosis, and necroptosis), destruction of intracellular organelles, and signaling pathway. The current therapeutic approaches have limited efficacy. Inhibition of RAS and use of antioxidants and antidiabetic drugs, such as inhibitors of sodium-glucose cotransporter 2 and dipeptidyl peptidase-4, have recently gained attention as therapeutic strategies to prevent renal scarring. This literature review highlights the state of the art regarding the molecular mechanisms relevant to the management of renal fibrosis caused by UUO.

Toll-interacting protein inhibits transforming growth factor beta signaling in mouse lung fibroblasts.

Variations in the Toll-interacting protein (TOLLIP) gene have been identified in genome-wide association studies to correlate with risk of disease, mortality, and response to N-acetylcysteine therapy in idiopathic pulmonary fibrosis. Although TOLLIP is known to modulate innate immune responses, its relevance in organ fibrogenesis remains unknown. Prior work in the literature suggests TOLLIP dampens transforming growth factor beta (TGFβ) signaling in human cell lines. In this study, we examined the role of TOLLIP in mouse lung fibroblast (MLF) responses to TGFβ and in the bleomycin model of experimental lung fibrosis using Tollip-/- mice. We hypothesize that if TOLLIP negatively regulates TGFβ signaling, then Tollip-/- mouse lung fibroblasts (MLFs) would have enhanced response to TGFβ treatment, and Tollip-/- mice would develop increased fibrosis following bleomycin challenge. Primary MLFs were stimulated with TGFβ (1 ng/mL) for 24 h. RNA was obtained to assess global transcriptional responses by RNA-seq and markers of myofibroblast transition by qPCR. Functional assessment of TGFβ-stimulated MLFs included cell migration by scratch assay, cell proliferation, and matrix invasion through Matrigel. In the invivo model of lung fibrosis, Tollip-/- mice and wild-type (WT) littermates were administered bleomycin intratracheally and assessed for fibrosis. We further examined TGFβ signaling invivo after bleomycin injury by SMAD2, ERK1/2, and TGFβR1 Western blot. In response to TGFβ treatment, both WT and Tollip-/- MLFs exhibited global transcriptional changes consistent with myofibroblast differentiation. However, Tollip-/- MLFs showed greater number of differentially expressed genes compared to WT MLFs and greater upregulation of Acta2 by qPCR. Functionally, Tollip-/- MLFs also exhibited increased migration and Matrigel invasiveness compared to WT. We found evidence of enhanced TGFβ signaling in Tollip-/- through SMAD2 invitro and invivo. Tollip-/- mice experienced lower survival using a standard weight-adjusted dosing without evidence of differences in fibrosis at Day 21. With adjustment of dosing for sex, no differences were observed in fibrosis at Day 21. However, Tollip-/- mice had greater weight loss and increased bronchoalveolar lavage fluid total protein during early resolution at Day 14 compared to WT without evidence of differences in acute lung injury at Day 7, suggesting impaired resolution of lung injury.

A novel BRD4 degrader, ARV-825, attenuates lung fibrosis through senolysis and antifibrotic effect

BackgroundRecent studies suggest that cellular senescence is related to the pathogenesis of idiopathic pulmonary fibrosis. However, cellular senescence has yet to be targeted therapeutically in clinical practice. ARV825, a recently developed BRD4 degrader, has been reported as a novel senolytic drug. Conversely, it has also been reported that BRD4 regulates the pro-fibrotic gene expression of fibroblasts. Therefore, this study focuses on the senolytic and anti-fibrotic effects of ARV825 and evaluated these effects on lung fibrosis. MethodsLung fibroblasts were induced to senescence through serial passage. The expression of senescence markers and pro-fibrotic markers were determined through quantitative PCR or immunoblot analysis. Lung fibrosis was induced in mice through intratracheal administration of bleomycin. Mice treated with ARV825 underwent histological analysis of lung fibrosis using the Ashcroft score. Total lung collagen was quantified through a hydroxyproline assay. Respiratory mechanics analysis was performed using the flexiVent system. ResultsFor senescent cells, ARV825 induced the expression of an apoptosis marker while reducing the expression of BRD4 and senescence markers. On the other hand, for early passage pre-senescent cells, ARV825 reduced the expression of collagen type 1 and α-smooth muscle actin. In an experimental mouse model of lung fibrosis, ARV825 attenuated lung fibrosis and improved lung function. Immunohistochemical staining revealed a significant decrease in the number of senescent alveolar type 2 cells in lung tissue due to ARV825 treatment. ConclusionsThese results suggest that ARV825 may impact the progressive and irreversible course of fibrotic lung diseases.

Intravenous immunoglobulins improve skin fibrosis in experimental models of systemic sclerosis

Systemic sclerosis (SSc) is the most severe systemic autoimmune disease with currently no cure. Intravenous immunoglobulins (IVIg) are an attractive candidate in this disease to counteract inflammation and fibrosis but data are scarce and conflicting. This study, assessed the effects of IVIg in a murine HOCl-induced model of SSc. We showed that IVIg prevented skin inflammation and fibrosis, by mitigating the immune cell infiltration (p = 0.04), proinflammatory cytokines gene overexpression (IL1β, p = 0.04; TNFα, p = 0.04; IL6, p = 0.05), skin and dermal thickening (p = 0.003 at d21 and p = 0.0003 at d42), the expression markers of fibrosis, such as αSMA (p = 0.031 for mRNA and p = 0.05 for protein), collagen (p = 0.05 for mRNA and p = 0.04 for protein, p = 0.05 for the hydroxyproline content) and fibronectin (p = 0.033 for mRNA). Moreover, IVIg prevented HOCl-induced alterations in splenic cell homeostasis. When administered in curative mode, despite their ability to reduce skin and dermal thickness (p < 0.0001 and p = 0.0002), IVIg showed partial or more mixed effects on skin inflammation and established fibrosis. These data favor further clinical trials in SSc patients on the potential efficiency of early and/or repeated IVIg administration.

Reciprocal regulation between the molecular clock and kidney injury.

Tubulointerstitial fibrosis is the common pathological substrate for many etiologies leading to chronic kidney disease. Although perturbations in the circadian rhythm have been associated with renal disease, the role of the molecular clock in the pathogenesis of fibrosis remains incompletely understood. We investigated the relationship between the molecular clock and renal damage in experimental models of injury and fibrosis (unilateral ureteral obstruction, folic acid, and adenine nephrotoxicity), using genetically modified mice with selective deficiencies of the clock components Bmal1, Clock, and Cry We found that the molecular clock pathway was enriched in damaged tubular epithelial cells with marked metabolic alterations. In human tubular epithelial cells, TGFβ significantly altered the expression of clock components. Although Clock played a role in the macrophage-mediated inflammatory response, the combined absence of Cry1 and Cry2 was critical for the recruitment of neutrophils, correlating with a worsening of fibrosis and with a major shift in the expression of metabolism-related genes. These results support that renal damage disrupts the kidney peripheral molecular clock, which in turn promotes metabolic derangement linked to inflammatory and fibrotic responses.

Physical Activity Protocols in Non-Alcoholic Fatty Liver Disease Management: A Systematic Review of Randomized Clinical Trials and Animal Models.

Non-alcoholic fatty liver disease (NAFLD) is closely associated with other metabolic disease and cardiovascular disease. Regular exercise reduces hepatic fat content and could be the first-line treatment in the management of NAFLD. This review aims to summarize the current evidence of the beneficial effects of exercise training and identify the molecular pathways involved in the response to exercise to define their role in the resolution of NAFLD both in animal and human studies. According to the inclusion criteria, 43 animal studies and 14 RCTs were included in this systematic review. Several exercise modalities were demonstrated to have a positive effect on liver function. Physical activity showed a strong association with improvement in inflammation, and reduction in steatohepatitis and fibrosis in experimental models. Furthermore, both aerobic and resistance exercise in human studies were demonstrated to reduce liver fat, and to improve insulin resistance and blood lipids, regardless of weight loss, although aerobic exercises may be more effective. Resistance exercise is more feasible for patients with NAFLD with poor cardiorespiratory fitness. More effort and awareness should be dedicated to encouraging NAFLD patients to adopt an active lifestyle and benefit from it its effects in order to reduce this growing public health problem.

The impact of hypoxia-inducible factors in the pathogenesis of kidney diseases: a link through cell metabolism.

Kidneys are sensitive to disturbances in oxygen homeostasis. Hypoxia and activation of the hypoxia-inducible factor (HIF) pathway alter the expression of genes involved in the metabolism of renal and immune cells, interfering with their functioning. Whether the transcriptional activity of HIF protects the kidneys or participates in the pathogenesis of renal diseases is unclear. Several studies have indicated that HIF signaling promotes fibrosis in experimental models of kidney disease. Other reports showed a protective effect of HIF activation on kidney inflammation and injury. In addition to the direct effect of HIF on the kidneys, experimental evidence indicates that HIF-mediated metabolic shift activates inflammatory cells, supporting the HIF cascade as a link between lung or gut damage and worsening of renal disease. Although hypoxia and HIF activation are present in several scenarios of renal diseases, further investigations are needed to clarify whether interfering with the HIF pathway is beneficial in different pathological contexts.

A polo-like kinase inhibitor identified by computational repositioning attenuates pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease with few effective therapeutic options. Recently, drug repositioning, which involves identifying novel therapeutic potentials for existing drugs, has been popularized as a new approach for the development of novel therapeutic reagents. However, this approach has not yet been fully utilized in the field of pulmonary fibrosis.MethodsThe present study identified novel therapeutic options for pulmonary fibrosis using a systematic computational approach for drug repositioning based on integration of public gene expression signatures of drug and diseases (in silico screening approach).ResultsAmong the top compounds predicted to be therapeutic for IPF by the in silico approach, we selected BI2536, a polo-like kinase (PLK) 1/2 inhibitor, as a candidate for treating pulmonary fibrosis using an in silico analysis. However, BI2536 accelerated mortality and weight loss rate in an experimental mouse model of pulmonary fibrosis. Because immunofluorescence staining revealed that PLK1 expression was dominant in myofibroblasts while PLK2 expression was dominant in lung epithelial cells, we next focused on the anti-fibrotic effect of the selective PLK1 inhibitor GSK461364. Consequently, GSK461364 attenuated pulmonary fibrosis with acceptable mortality and weight loss in mice.ConclusionsThese findings suggest that targeting PLK1 may be a novel therapeutic approach for pulmonary fibrosis by inhibiting lung fibroblast proliferation without affecting lung epithelial cells. In addition, while in silico screening is useful, it is essential to fully determine the biological activities of candidates by wet-lab validation studies.

Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells

The cellular (centralized) communication network (CCN) factor protein family contains six small secreted cysteine-rich proteins sharing high structural similarity. These matricellular proteins have vital biological functions in cell adhesion, migration, cell cycle progression, and control of production and degradation of extracellular matrix. However, in liver the biological functions of CCN proteins become most visible during hepatic injury, disease, and remodeling. In particular, most of the hepatic functions of CCN proteins were derived from CCN2/CTGF, which becomes highly expressed in damaged hepatocytes and acts as a profibrogenic molecule. On the contrary, CCN1/CYR61 seems to have opposite effects, while the biological activity during hepatic fibrosis is somewhat controversially discussed for other CCN family members. In the present study, we analyzed the expression of CCN5/WISP2 in cultures of different types of primary liver cells and in an experimental model of hepatic fibrosis. We found that CCN5 is expressed in hepatic stellate cells, myofibroblasts and portal myofibroblasts, while CCN5 expression is virtually absent in hepatocytes. During hepatic fibrogenesis, CCN5 is significantly upregulated. Overexpression of CCN5 in portal myofibroblasts reduced expression of transforming growth factor-β receptor I (ALK5) and concomitant Smad2 activation, whereas JunB expression is upregulated. Moreover, elevated expression of CCN5 induces endoplasmic reticulum stress, unfolded protein response and apoptosis in portal myofibroblasts. We suggest that upregulated expression of CCN5 might be an intrinsic control mechanism that counteracts overshooting fibrotic responses in profibrogenic liver cells.Graphical abstract

Interleukin 36 receptor-inducible matrix metalloproteinase 13 mediates intestinal fibrosis.

Fibrostenotic disease is a common complication in Crohn's disease (CD) patients hallmarked by transmural extracellular matrix (ECM) accumulation in the intestinal wall. The prevention and medical therapy of fibrostenotic CD is an unmet high clinical need. Although targeting IL36R signaling is a promising therapy option, downstream mediators of IL36 during inflammation and fibrosis have been incompletely understood. Candidate molecules include matrix metalloproteinases which mediate ECM turnover and are thereby potential targets for anti-fibrotic treatment. Here, we have focused on understanding the role of MMP13 during intestinal fibrosis. We performed bulk RNA sequencing of paired colon biopsies taken from non-stenotic and stenotic areas of patients with CD. Corresponding tissue samples from healthy controls and CD patients with stenosis were used for immunofluorescent (IF) staining. MMP13 gene expression was analyzed in cDNA of intestinal biopsies from healthy controls and in subpopulations of patients with CD in the IBDome cohort. In addition, gene regulation on RNA and protein level was studied in colon tissue and primary intestinal fibroblasts from mice upon IL36R activation or blockade. Finally, in vivo studies were performed with MMP13 deficient mice and littermate controls in an experimental model of intestinal fibrosis. Ex vivo tissue analysis included Masson's Trichrome and Sirius Red staining as well as evaluation of immune cells, fibroblasts and collagen VI by IF analysis. Bulk RNA sequencing revealed high upregulation of MMP13 in colon biopsies from stenotic areas, as compared to non-stenotic regions of patients with CD. IF analysis confirmed higher levels of MMP13 in stenotic tissue sections of CD patients and demonstrated αSMA+ and Pdpn+ fibroblasts as a major source. Mechanistic experiments demonstrated that MMP13 expression was regulated by IL36R signaling. Finally, MMP13 deficient mice, as compared to littermate controls, developed less fibrosis in the chronic DSS model and showed reduced numbers of αSMA+ fibroblasts. These findings are consistent with a model suggesting a molecular axis involving IL36R activation in gut resident fibroblasts and MMP13 expression during the pathogenesis of intestinal fibrosis. Targeting IL36R-inducible MMP13 could evolve as a promising approach to interfere with the development and progression of intestinal fibrosis.