Fibrosis In Multiple Organs Research Articles

LncRNA H19 accelerates renal fibrosis by negatively regulating the let-7b-5p/TGF-βR1/COL1A1 axis

BackgroundTransforming growth factor-beta1 (TGF-β1)-mediated renal fibrosis is a critical pathological process of chronic kidney disease worsening to end-stage renal disease. Recent studies have shown that long noncoding RNA H19 (lncRNA H19) is widely involved in the formation and progression of fibrosis in multiple organs. However, its molecular events in renal fibrosis remain to be elucidated. MethodsRats were treated with adenine intragastrically and HK-2 cells were induced by TGF-β1 to construct renal fibrosis models in vivo and in vitro, respectively. Renal histopathological examination was performed using HE and Masson staining. Gene expression levels of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), TGF-β1, fibronectin (Fn), alpha-smooth muscle actin (α-SMA), H19, let-7b-5p, TGF-β receptor 1 (TGF-βR1), and type I collagen (COL1A1) were detected by qRT-PCR. Immunohistochemistry, immunofluorescence, and western blot analysis were used to evaluate the expression of renal fibrosis biomarkers. Dual-luciferase reporter assay was used to verify the presence of binding sites between H19 and let-7b-5p, and between let-7b-5p and TGF-βR1 and COL1A1. ResultsH19 was overexpressed in both in vivo and in vitro renal fibrosis models. H19 knockdown significantly reversed TGF-β1-induced upregulation of fibronectin, COL1A1, and α-SMA and downregulation of E-cadherin in HK-2 cells, accompanied by an increase in let-7b-5p. Let-7b-5p was bound to H19 in HK-2 cells, and its overexpression inhibited TGF-β1-induced HK-2 cell fibrosis. Further experiments determined that let-7b-5p directly targets TGF-βR1 and COL1A1 in HK-2 cells. In addition, inhibition of let-7b-5p reversed the reduction in HK-2 cell fibrosis induced by H19 knockdown. Finally, knockdown of H19 alleviated renal fibrosis in vivo and was associated with regulation of the let-7b-5p/TGF-βR1/COL1A1 axis. ConclusionOur results indicate that knockdown of H19 inhibits renal tubular epithelial fibrosis by negatively regulating the let-7b-5p/TGF-βR1/COL1A1 axis, which may provide new mechanistic insights into CRF progression.

The impact of psoriasis on idiopathic pulmonary fibrosis: atwo-sample Mendelian randomization study.

The association between psoriasis and pulmonary fibrosis has been reported in observational studies. However, the association is vulnerable to bias from using immunosuppressants such as methotrexate, which can cause fibrosis in multiple organs. The present study is to investigate whether psoriasis could independently increase the risk of idiopathic pulmonary fibrosis (IPF). We conducted a two-sample Mendelian randomization (MR) study using summary statistics from genome-wide association studies. The random-effects inverse variance weighted analysis was used as the primary method. Some secondary analyses were further performed, including a sensitivity analysis using a genetic instrument that excluded extended single nucleotide polymorphisms (SNPs) in the major histocompatibility complex (MHC) gene region. Our study included 9267 cases and 364,071 controls for psoriasis, 2018 cases, and 373,064 controls for IPF of European ancestry, respectively. Genetically predicted psoriasis was associated with a higher risk of IPF (odds ratio (OR), 1.14; 95% confidence interval (CI), 1.08-1.22; P < 0.001). Sensitivity analyses did not uncover any statistically significant evidence of bias resulting from pleiotropy or the genetic instruments, including SNPs in the MHC gene region. These MR analyses support that genetically predicted psoriasis was associated with the risk of IPF, implying that pulmonary fibrosis in patients with psoriasis should not be neglected, even if they are not receiving immunosuppressant therapy.

Whole-exome sequencing revealed a novel mutation of the ALMS1 gene in a Chinese family with Alström syndrome: a case report

BackgroundAlström syndrome (AS) is a rare autosomal recessive disorder that leads to multiple organ fibrosis and failure. Precise diagnosis from the clinical symptoms is challenging due to its highly variabilities and its frequent confusion with other ciliopathies and genetic diseases. Currently, mutations in the ALMS1 gene have been reported as a major cause of AS, thus, it is crucial to focus on the detection and discovery of ALMS1 mutations.Case presentationWe present a case of a 13-year-old Chinese boy weighing 70 kg and standing 168 cm tall. He has two younger brothers. Their parents hail from different ancestral homes in eastern and northern China. The patient’s primary clinical findings included visual impairment at the age of four and progressive hearing loss starting at the age of ten. Subsequently, at the age of twelve, the patient developed hyperlipidaemia and hyperinsulinemia. Ultrasonographic findings indicated the presence of gallstones and mild fatty liver. His Body Mass Index (BMI) significantly increased to 25 kg/m2 (ref: 18.5–23.9 kg/m2). Additionally, echocardiography revealed mild mitral and tricuspid regurgitation. Ultimately, Whole Exome Sequencing (WES) identified a new missense mutation in the ALMS1 gene (NG_011690.1 (NM_015120): c.9536G > A (p.R3179Q)). This missense mutation generated an aberrant splicer and disrupted the stability and hydrophobicity of proteins, which preliminarily determined as “ likely pathogenic”. Therefore, considering all the above symptoms and molecular analysis, we deduced that the patient was diagnosed with AS according to the guidelines. We recommended that he continue wearing glasses and undergo an annual physical examination.ConclusionIn this case report, we report a novel homozygous ALMS1 mutation associated with AS in the Chinese population, which expands the mutation spectrum of ALMS1. Genetic testing indeed should be incorporated into the diagnosis of syndromic deafness, as it can help avoid misdiagnoses of AS. While there is no specific treatment for AS, early diagnosis and intervention can alleviate the progression of some symptoms and improve patients’ quality of life.

CTHRC1: An Emerging Hallmark of Pathogenic Fibroblasts in Lung Fibrosis.

Pulmonary fibrosis is a chronic, progressive, irreversible lung disease characterized by fibrotic scarring in the lung parenchyma. This condition involves the excessive accumulation of extracellular matrix (ECM) due to the aberrant activation of myofibroblasts in the alveolar environment. Transforming growth factor beta (TGF-β) signaling is a crucial driver of fibrogenesis because it promotes excessive ECM deposition, thereby leading to scar formation and lung damage. A primary target of TGF-β signaling in fibrosis is Collagen Triple Helix Repeat Containing 1 (CTHRC1), a secreted glycoprotein that plays a pivotal role in ECM deposition and wound repair. TGF-β transcriptionally regulates CTHRC1 in response to tissue injury and controls the wound healing response through functional activity. CTHRC1 may also play an essential role in re-establishing and maintaining tissue homeostasis after wound closure by modulating both the TGF-β and canonical Wnt signaling pathways. This dual function suggests that CTHRC1 regulates tissue remodeling and homeostasis. However, deregulated CTHRC1 expression in pathogenic fibroblasts has recently emerged as a hallmark of fibrosis in multiple organs and tissues. This review highlights recent studies suggesting that CTHRC1 can serve as a diagnostic and prognostic biomarker for fibrosis in idiopathic pulmonary fibrosis, systemic sclerosis, and post-COVID-19 lung fibrosis. Notably, CTHRC1 expression is responsive to antifibrotic drugs that target the TGF-β pathway, such as pirfenidone and bexotegrast, indicating its potential as a biomarker of treatment success. These findings suggest that CTHRC1 may present new opportunities for diagnosing and treating patients with lung fibrosis.

Galectin-receptor interaction: a key player in liver fibrosis induced by Schistosoma japonicum infection

BackgroundSchistosoma japonicum eggs lodge in the liver and induce a fibrotic granulomatous immune response in the liver of host. Galectin 3 (Gal-3) is a protein implicated in fibrosis in multiple organs. However, the pathology and molecular mechanisms promoting hepatic granuloma formation remain poorly understood.MethodsTo investigate the effect of blocking galectin-receptor interactions by α-lactose on liver immunopathology in mice with S. japonicum infection, C57BL/6 mice were infected with S. japonicum and alpha (α)-lactose was intraperitoneally injected to block the interactions of galectins and their receptors.ResultsCompared with S. japonicum-infected mice, there were significantly decreased Gal-3 mRNA and protein expression levels, decreased intensity of Gal-3 fluorescence in the liver, decreased serum ALT and AST levels, decreased egg numbers of S. japonicum in the liver section, attenuated hepatic and spleen pathology, and alleviated liver fibrosis accompanied with decreased protein expression levels of fibrosis markers [α-smooth muscle actin (α-SMA), collagen I, and collagen IV] in the liver of S. japonicum-infected mice blocked galectin-receptor interactions with hematoxylin-eosin staining, Masson’s trichrome staining, immunohistochemistry, or Western blot analysis. Compared with S. japonicum-infected mice, blocking galectin-receptor interactions led to increased eosinophil infiltration and higher eosinophil cationic protein (ECP) expression in the liver, accompanied by increased mRNA levels of eosinophil granule proteins [ECP and eosinophil peroxidase (EPO)], IL-5, CCL11, and CCR3 in the liver and decreased mRNA levels of Gal-3 and M2 macrophage cytokines (TGF-β, IL-10, and IL-4) in the liver and spleen by using quantitative real-time reverse transcription-polymerase chain reaction. In addition, there were increased Beclin1 protein expression and protein expression ratio of LC3B-II/LC3B-I and decreased p62 protein expression and protein expression ratios of phospho-mTOR/mTOR and phospho-AKT/AKT by Western blot; increased double-labeled F4/80+/LC3B+ cells by immunofluorescence staining; increased M1 macrophage polarization in the liver of S. japonicum-infected mice blocked galectin-receptor interactions by flow cytometric analysis and immunofluorescence staining.ConclusionsOur data found that blockage of galectin-receptor interactions downregulated Gal-3, which in turn led to reduced liver functional damage, elevated liver eosinophil recruitment, promoted macrophage autophagy through the Akt/mTOR signaling pathway, and alleviated liver pathology and fibrosis. Therefore, Gal-3 plays a pivotal role during S. japonicum infection and could be a target of pharmacologic potential for liver fibrosis induced by S. japonicum infection.Graphical

Inhibition of MERTK reduces organ fibrosis in mouse models of fibrotic disease.

Transforming growth factor-β (TGFβ) drives fibrosis and disease progression in a number of chronic disorders, but targeting this ubiquitously expressed cytokine may not yield a viable and safe antifibrotic therapy. Here, we sought to identify alternative ways to inhibit TGFβ signaling using human hepatic stellate cells and macrophages from humans and mice in vitro, as well as mouse models of liver, kidney, and lung fibrosis. We identified Mer tyrosine kinase (MERTK) as a TGFβ-inducible effector of fibrosis that was up-regulated during fibrosis in multiple organs in three mouse models. We confirmed these findings in liver biopsy samples from patients with metabolic dysfunction-associated fatty liver disease (MAFLD). MERTK also induced TGFβ expression and drove TGFβ signaling resulting in a positive feedback loop that promoted fibrosis in cultured cells. MERTK regulated both canonical and noncanonical TGFβ signaling in both mouse and human cells in vitro. MERTK increased transcription of genes regulating fibrosis by modulating chromatin accessibility and RNA polymerase II activity. In each of the three mouse models, disrupting the fibrosis-promoting signaling loop by reducing MERTK expression reduced organ fibrosis. Pharmacological inhibition of MERTK reduced fibrosis in these mouse models either when initiated immediately after injury or when initiated after fibrosis was established. Together, these data suggest that MERTK plays a role in modulating organ fibrosis and may be a potential target for treating fibrotic diseases.

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.

Endothelial cells drive organ fibrosis in mice by inducing expression of the transcription factor SOX9.

Fibrosis is a hallmark of chronic disease. Although fibroblasts are involved, it is unclear to what extent endothelial cells also might contribute. We detected increased expression of the transcription factor Sox9 in endothelial cells in several different mouse fibrosis models. These models included systolic heart failure induced by pressure overload, diastolic heart failure induced by high-fat diet and nitric oxide synthase inhibition, pulmonary fibrosis induced by bleomycin treatment, and liver fibrosis due to a choline-deficient diet. We also observed up-regulation of endothelial SOX9 in cardiac tissue from patients with heart failure. To test whether SOX9 induction was sufficient to cause disease, we generated mice with endothelial cell-specific overexpression of Sox9, which promoted fibrosis in multiple organs and resulted in signs of heart failure. Endothelial Sox9 deletion prevented fibrosis and organ dysfunction in the two mouse models of heart failure as well as in the lung and liver fibrosis mouse models. Bulk and single-cell RNA sequencing of mouse endothelial cells across multiple vascular beds revealed that SOX9 induced extracellular matrix, growth factor, and inflammatory gene expression, leading to matrix deposition by endothelial cells. Moreover, mouse endothelial cells activated neighboring fibroblasts that then migrated and deposited matrix in response to SOX9, a process partly mediated by the secreted growth factor CCN2, a direct SOX9 target; endothelial cell-specific Sox9 deletion reversed these changes. These findings suggest a role for endothelial SOX9 as a fibrosis-promoting factor in different mouse organs during disease and imply that endothelial cells are an important regulator of fibrosis.

Targeted inhibition of transforming growth factor-β type I receptor by AZ12601011 improves paraquat poisoning-induced multiple organ fibrosis

Paraquat (PQ) causes fatal poisoning that leads to systemic multiple organ fibrosis, and transforming growth factor (TGF)-β1 plays a critical role in this process. In this study, we aimed to investigate the effects of AZ12601011 (a small molecular inhibitor of TGFβRI) on PQ-induced multiple organ fibrosis. We established a mouse model of PQ in vivo and used PQ-treated lung epithelial cell (A549) and renal tubular epithelial cells (TECs) in vitro. Haematoxylin–eosin and Masson staining revealed that AZ12601011 ameliorated pulmonary, hepatic, and renal fibrosis, consistent with the decrease in the levels of fibrotic indicators, alpha-smooth muscle actin (α-SMA) and collagen-1, in the lungs and kidneys of PQ-treated mice. In vitro data showed that AZ12601011 suppressed the induction of α-SMA and collagen-1 in PQ-treated A549 cells and TECs. In addition, AZ12601011 inhibited the release of inflammatory factors, interleukin (IL)-1β, IL-6, and tumour necrosis factor-α. Mechanistically, TGF-β and TGFβRI levels were significantly upregulated in the lungs and kidneys of PQ-treated mice. Cellular thermal shift assay and western blotting revealed that AZ12601011 directly bound with TGFβRI and blocked the activation of Smad3 downstream. In conclusion, our findings revealed that AZ12601011 attenuated PQ-induced multiple organ fibrosis by blocking the TGF-β/Smad3 signalling pathway, suggesting its potential for PQ poisoning treatment.

Heavy-chain antibody targeting of CD38 NAD+ hydrolase ectoenzyme to prevent fibrosis in multiple organs

The functionally pleiotropic ectoenzyme CD38 is a glycohydrolase widely expressed on immune and non-hematopoietic cells. By converting NAD+ to ADP-ribose and nicotinamide, CD38 governs organismal NAD+ homeostasis and the activity of NAD+-dependent cellular enzymes. CD38 has emerged as a major driver of age-related NAD+ decline underlying adverse metabolic states, frailty and reduced health span. CD38 is upregulated in systemic sclerosis (SSc), a chronic disease characterized by fibrosis in multiple organs. We sought to test the hypothesis that inhibition of the CD38 ecto-enzymatic activity using a heavy-chain monoclonal antibody Ab68 will, via augmenting organismal NAD+, prevent fibrosis in a mouse model of SSc characterized by NAD+ depletion. Here we show that treatment of mice with a non-cytotoxic heavy-chain antibody that selectively inhibits CD38 ectoenzyme resulted in NAD+ boosting that was associated with significant protection from fibrosis in multiple organs. These findings suggest that targeted inhibition of CD38 ecto-enzymatic activity could be a potential pharmacological approach for SSc fibrosis treatment.

Abstract 15180: Inhibition of Tgfβ Signaling Enables Durable Ventricular Pacing by TBX18 Gene Transfer

Introduction: Somatic gene transfer of an embryonic transcription factor, TBX18, could directly reprogram ventricular myocytes to induced pacemaker myocytes. We and others have previously reported that direct myocardial gene delivery of TBX18 created ventricular pacing in heart block animals in vivo. However, the de novo biological pacing was short-lived, diminishing its translational value. Hypothesis: Our preliminary RNAseq and mass spec data indicate pronounced Tgfβ signaling in TBX18-transduced cardiomyocytes. Tgfβ signaling induces fibrosis in multiple organs including the heart. We hypothesized that TBX18 triggers Tgfβ signaling and results in fibrosis, leading to loss of durable ventricular pacing. Methods: Adenoviral vectors expressing TBX18 or GFP was delivered to neonatal rat ventricular myocytes (NRVMs) in vitro or to the left ventricular apex of adult rats with complete atrioventricular block (CAVB). Results: In vitro experiments showed increased population of vimentin+ fibroblasts and alpha smooth muscle actin+ myofibroblasts in TBX18-NRVMs compared to GFP-NRVMs at 2 weeks after gene transfer. Single cell transcriptome data indicated that TBX18 triggered substantial changes in extracellular matrix-related gene expression and Tgfβ signaling. Quantitative RT-PCR and ELISA validated scRNA-seq data, indicating fibroblast activation. TBX18-NRVMs showed faster automaticity than that control did, but their automaticity began wavering after day 7. Treatment of TBX18-NRVMs with a Tgfβ signaling inhibitor, A83-01, mitigated fibroblast activation by TBX18 and preserved their automaticity. Direct delivery of TBX18 into ventricular myocardium resulted in focal fibrosis as well as expansion of vimentin+ fibroblasts at day 7. TBX18 gene transfer initiated ventricular pacing in a rat model of CAVB, but ventricular pacing diminished after one week. In contrast, treating TBX18-injected animals with A83-01 enabled sustained biological pacing beyond three weeks of after gene delivery, accompanied by reduced fibrosis in situ. Conclusions: Inhibition of Tgfβ signaling is sufficient to achieve durable cardiac pacing by TBX18-induced biological pacemakers.

THU575 The Canonical Wnt/β-catenin Pathway Is Not Involved In Estrogen-induced Skeletal Muscle Fibrosis In A Mouse Model Of Inguinal Hernia

Abstract Disclosure: T. You: None. H. Zhao: None. T. Potluri: None. P. Yin: None. S.E. Bulun: None. Inguinal hernias affect up to 27% of elderly men, with approximately 800,000 inguinal hernia repair surgeries performed annually. While these procedures are often effective, a significant subset of hernias are refractory to treatment, resulting in high mortality, high recurrence, and chronic pain. Until recently, little research has been done examining the molecular mechanisms that drive hernia-associated lower abdominal wall weakening. Our lab previously created a transgenic mouse model expressing the human aromatase gene (Aromhum) and showed that male Aromhum mice produced high levels of estrogen in the lower abdominal muscle (LAM) tissue, leading to extensive LAM fibrosis, muscle atrophy, and hernia formation. Single-cell analysis of fibrotic LAM tissue in these mice showed high expression of estrogen receptor alpha (ERα) in LAM fibroblasts as well as upregulation of Wnt signaling pathways in these ERα-positive fibroblasts. The canonical Wnt/β-catenin signaling pathway has been implicated in fibroblast activation and fibrosis in multiple organs, including the lung, heart, and skin. Thus, we hypothesized that canonical Wnt/β-catenin signaling acts as a downstream mediator of ERα to cause LAM fibroblast activation, tissue fibrosis, and hernia. To test our hypothesis, we cultured primary ERα-positive LAM fibroblasts from Aromhum mice in the presence of estrogen with or without siRNA knockdown (KD) of β-catenin. However, KD of β-catenin under these conditions did not alter fibroblast proliferation as measured by EdU assay, nor did it alter expression of genes involved in fibroblast activation or extracellular matrix formation. Furthermore, administration of LGK974, a Wnt signaling inhibitor, in male Aromhum mice did not prevent hernia development, decrease hernia size, or decrease the extent of LAM fibrosis and muscle atrophy. Thus, we concluded that the canonical Wnt/β-catenin signaling pathway does not serve as a downstream mediator of estrogen-induced skeletal muscle fibrosis in our Aromhum mouse model of inguinal hernia. Further studies are needed to evaluate the role of Wnt/β-catenin signaling in other inguinal hernia models or in humans. Presentation Date: Thursday, June 15, 2023

Loss of endothelial glucocorticoid receptor accelerates organ fibrosis in db/db mice.

Endothelial cells play a key role in maintaining homeostasis and are deranged in many disease processes, including fibrotic conditions. Absence of the endothelial glucocorticoid receptor (GR) has been shown to accelerate diabetic kidney fibrosis in part through upregulation of Wnt signaling. The db/db mouse model is a model of spontaneous type 2 diabetes that has been noted to develop fibrosis in multiple organs over time, including the kidneys. This study aimed to determine the effect of loss of endothelial GR on organ fibrosis in the db/db model. db/db mice lacking endothelial GR showed more severe fibrosis in multiple organs compared with endothelial GR-replete db/db mice. Organ fibrosis could be substantially improved either through administration of a Wnt inhibitor or metformin. IL-6 is a key cytokine driving the fibrosis phenotype and is mechanistically linked to Wnt signaling. The db/db model is an important tool to study the mechanisms of fibrosis and its phenotype in the absence of endothelial GR highlights the synergistic effects of Wnt signaling and inflammation in the pathogenesis or organ fibrosis.NEW & NOTEWORTHY The major finding of this work is that endothelial glucocorticoid receptor-mediated upregulation of Wnt signaling and concurrent hyperinflammation work synergistically to exacerbate organ fibrosis in a genetic mouse model of diabetes. This study adds to our understanding of diabetic renal fibrosis and has important implications for the use of metformin in this condition.

Interleukin-13 contributes to the occurrence of oral submucosal fibrosis.

Oral submucous fibrosis (OSF) is a chronic progressive fibrosis disease that affects in oral mucosal tissues. Interleukin (IL)-13 has been implicated in the development of fibrosis in multiple organs. Indeed, it contributes to diseases such as pulmonary fibrosis, liver cirrhosis among others. Currently, its expression in OSF and the specific mechanisms are not well understood. The aim of this study was to investigate the role of IL-13 in OSF and further explore whether IL-13 regulates-polarization of M2-macrophages in OSF. Initially, in the tissues of patients with OSF, we observed a high expression of M2-macrophages and IL-13 protein. Additionally, we found a correlation between the expression of IL-13 and the stage of OSF. Arecoline inhibited the proliferation of fibroblasts (FBs) and promoted IL-13 production in vitro. Furthermore, our observations revealed that M2-macrophages increased upon co-culturing M0-macrophages with supernatants containing the IL-13 cytokine. In conclusion, our study demonstrated that arecoline stimulates FBs leading to increased secretion of IL-13, which in turn IL-13 leads to polarization of M2-macrophages and promotes the occurrence of OSF. This suggests that IL-13 may be a potential therapeutic target of OSF.

Nintedanib prevents TGF-β2-induced epithelial-mesenchymal transition in retinal pigment epithelial cells

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a key fibrosis pathogenesis in proliferative vitreoretinopathy (PVR). However, few medicines can prevent proliferative membranes and cell proliferation in the clinic. Nintedanib, a tyrosine kinase inhibitor, has been shown to prevent fibrosis and be anti-inflammatory in multiple organ fibrosis. In our study, 0.1, 1, 10 μM nintedanib was added to 20 ng/mL transforming growth factor beta 2 (TGF-β2)-induced EMT in ARPE-19 cells. Western blot and immunofluorescence assay showed that 1 μM nintedanib suppressed TGF-β2-induced E-cadherin expression decreased and Fibronectin, N-cadherin, Vimentin, and α-SMA expression increased. Quantitative real-time PCR results showed that 1 μM nintedanib decreased TGF-β2-induced increase in SNAI1, Vimentin, and Fibronectin expression and increased TGF-β2-induced decrease in E-cadherin expression. In addition, the CCK-8 assay, wound healing assay, and collagen gel contraction assay also showed that 1 μM nintedanib ameliorated TGF-β2-induced cell proliferation, migration, and contraction, respectively. These results suggested that nintedanib inhibits TGF-β2-induced EMT in ARPE-19 cells, which may be a potential pharmacological treatment for PVR.

Salvianolic Acid B Attenuates Hypertrophic Scar Formation In Vivo and In Vitro.

Hypertrophic scars (HTSs) are a fibroproliferative disorder that occur following skin injuries. Salvianolic acid B (Sal-B) is an extractant from Salvia miltiorrhiza that has been reported to ameliorate fibrosis in multiple organs. However, the antifibrotic effect on HTSs remains unclear. This study aimed to determine the antifibrotic effect of Sal-B in vitro and in vivo. In vitro, hypertrophic scar-derived fibroblasts (HSFs) were isolated from human HTSs and cultured. HSFs were treated with (0, 10, 50, 100 μmol/L) Sal-B. Cell proliferation and migration were evaluated by EdU, wound healing, and transwell assays. The protein and mRNA levels of TGFβI, Smad2, Smad3, α-SMA, COL1, and COL3 were detected by Western blots and real-time PCR. In vivo, tension stretching devices were fixed on incisions for HTS formation. The induced scars were treated with 100 μL of Sal-B/PBS per day according to the concentration of the group and followed up for 7 or 14 days. The scar condition, collagen deposition, and α-SMA expression were analyzed by gross visual examination, H&E, Masson, picrosirius red staining, and immunofluorescence. In vitro, Sal-B inhibited HSF proliferation, migration, and downregulated the expression of TGFβI, Smad2, Smad3, α-SMA, COL1, and COL3 in HSFs. In vivo, 50 and 100 μmol/L Sal-B significantly reduced scar size in gross and cross-sectional observations, with decreased α-SMA expression and collagen deposition in the tension-induced HTS model. Our study demonstrated that Sal-B inhibits HSFs proliferation, migration, fibrotic marker expression and attenuates HTS formation in a tension-induced HTS model in vivo. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Nuclear IL-33 in Fibroblasts Promotes Skin Fibrosis

Systemic sclerosis (SSc) is a complex and poorly understood connective tissue disease characterized by diffuse fibrosis in several organs, especially the skin ( Asano, 2017 Asano Y. Recent advances in the treatment of skin involvement in systemic sclerosis. Inflamm Regen. 2017; 37: 12 Crossref Scopus (6) Google Scholar , Bhattacharyya et al., 2012 Bhattacharyya S. Wei J. Tourtellotte W.G. Hinchcliff M. Gottardi C.G. Varga J. Fibrosis in systemic sclerosis: common and unique pathobiology. Fibrogenesis Tissue Repair. 2012; 5: S18 Crossref Scopus (30) Google Scholar , Denton et al., 2006 Denton C.P. Black C.M. Abraham D.J. Mechanisms and consequences of fibrosis in systemic sclerosis. Nat Clin Pract Rheumatol. 2006; 2: 134-144 Crossref Scopus (156) Google Scholar ). Interleukin (IL)-33 is found to regulate tissue fibrosis in multiple organs ( Kotsiou et al., 2018 Kotsiou O.S. Gourgoulianis K.I. Zarogiannis S.G. IL-33/ST2 Axis in Organ Fibrosis. Frontiers in immunology. 2018; 9: 2432 Crossref PubMed Scopus (96) Google Scholar , Xu et al., 2016 Xu J. Zheng J. Song P. Zhou Y. Guan S. IL33/ST2 pathway in a bleomycininduced pulmonary fibrosis model. Mol Med Rep. 2016; 14: 1704-1708 Crossref Scopus (13) Google Scholar ). However, the exact role of IL-33 in fibrosis remains controversial ( Kotsiou et al., 2018 Kotsiou O.S. Gourgoulianis K.I. Zarogiannis S.G. IL-33/ST2 Axis in Organ Fibrosis. Frontiers in immunology. 2018; 9: 2432 Crossref PubMed Scopus (96) Google Scholar ). Previous studies have shown a pro-fibrotic role for IL-33 cytokine through its receptor, interleukin-1 receptor-like 1(IL1RL1 or ST2), regulating the secretion of pro-fibrotic cytokine, IL-13, from eosinophils, M2 macrophages and type 2 innate lymphoid cells (ILC2) ( Li et al., 2014 Li D. Guabiraba R. Besnard A.G. Komai-Koma M. Jabir M.S. Zhang L. et al. IL-33 promotes ST2-dependent lung fibrosis by the induction of alternatively activated macrophages and innate lymphoid cells in mice. The Journal of allergy and clinical immunology. 2014; 134: 1422-1432 e11 Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar , Rankin et al., 2010 Rankin A.L. Mumm J.B. Murphy E. Turner S. Yu N. McClanahan T.K. et al. IL-33 induces IL-13-dependent cutaneous fibrosis. J Immunol. 2010; 184: 1526-1535 Crossref PubMed Scopus (188) Google Scholar , Wu et al., 2018 Wu L. Luo Z. Zheng J. Yao P. Yuan Z. Lv X. et al. IL-33 Can Promote the Process of Pulmonary Fibrosis by Inducing the Imbalance Between MMP-9 and TIMP-1. Inflammation. 2018; 41: 878-885 Crossref PubMed Scopus (30) Google Scholar ). However, IL-33 can also play an anti-fibrotic role through the induction of ILC2 cells and regulatory T cells in several organs including the skin ( Cheon et al., 2022 Cheon S.Y. Park J.H. Ameri A.H. Lee R.T. Nazarian R.M. Demehri S. IL-33/Regulatory T-Cell Axis Suppresses Skin Fibrosis. The Journal of investigative dermatology. 2022; Google Scholar , Garbern et al., 2019 Garbern J.C. Williams J. Kristl A.C. Malick A. Rachmin I. Gaeta B. et al. Dysregulation of IL-33/ST2 signaling and myocardial periarteriolar fibrosis. J Mol Cell Cardiol. 2019; 128: 179-186 Abstract Full Text Full Text PDF Scopus (12) Google Scholar , Riedel et al., 2017 Riedel J.H. Becker M. Kopp K. Duster M. Brix S.R. Meyer-Schwesinger C. et al. IL-33-Mediated Expansion of Type 2 Innate Lymphoid Cells Protects from Progressive Glomerulosclerosis. J Am Soc Nephrol. 2017; 28: 2068-2080 Crossref Scopus (71) Google Scholar ). Thus, it is critical to determine the full spectrum of IL-33 effect in fibrosis to develop optimal therapies for its utilization in the treatment of SSc and other fibrosis-associated diseases.

Targeting Mitochondrial Sirtuins in Age-Related Neurodegenerative Diseases and Fibrosis.

Aging is a natural and complex biological process that is associated with widespread functional declines in numerous physiological processes, terminally affecting multiple organs and tissues. Fibrosis and neurodegenerative diseases (NDs) often occur with aging, imposing large burdens on public health worldwide, and there are currently no effective treatment strategies for these diseases. Mitochondrial sirtuins (SIRT3-5), which are members of the sirtuin family of NAD+-dependent deacylases and ADP-ribosyltransferases, are capable of regulating mitochondrial function by modifying mitochondrial proteins that participate in the regulation of cell survival under various physiological and pathological conditions. A growing body of evidence has revealed that SIRT3-5 exert protective effects against fibrosis in multiple organs and tissues, including the heart, liver, and kidney. SIRT3-5 are also involved in multiple age-related NDs, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. Furthermore, SIRT3-5 have been noted as promising targets for antifibrotic therapies and the treatment of NDs. This review systematically highlights recent advances in knowledge regarding the role of SIRT3-5 in fibrosis and NDs and discusses SIRT3-5 as therapeutic targets for NDs and fibrosis.

Role of TGF-β Signaling in Coronavirus Disease 2019

Abstract Coronavirus disease 2019 (COVID-19) has a broad spectrum of clinical manifestations involving the respiratory, cardiovascular, renal, neuropsychiatric, gastrointestinal, and dermatological systems. Some patients with COVID-19 experience acute infection and post-COVID-19 syndrome. There is increasing evidence that TGF-β signaling plays an important role in the pathogenesis of both acute and chronic COVID-19 infection. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein was reported to interact with Smad3, a key downstream mediator of TGF-β signaling, thereby promoting TGF-β1/Smad3 signaling and causing cell death during the acute phase of COVID-19 infection. Because activation of TGF-β/Smad3 signaling has an essential role in multiple organ fibrosis, it is possible that overreactive TGF-β/Smad3 signaling may cause tissue fibrosis in the lung, heart, and kidney after SARS-CoV-2 infection. Thus, not only administration of antiviral drugs and traditional Chinese medicines, but also targeting of TGF-β signaling components, particularly Smad3, with various therapeutic strategies involving OT-101, pirfenidone, and specific Smad3 inhibitors, such as SIS3, may provide novel and specific therapies for COVID-19 patients.

Fibroblast A20 governs fibrosis susceptibility and its repression by DREAM promotes fibrosis in multiple organs

In addition to autoimmune and inflammatory diseases, variants of the TNFAIP3 gene encoding the ubiquitin-editing enzyme A20 are also associated with fibrosis in systemic sclerosis (SSc). However, it remains unclear how genetic factors contribute to SSc pathogenesis, and which cell types drive the disease due to SSc-specific genetic alterations. We therefore characterize the expression, function, and role of A20, and its negative transcriptional regulator DREAM, in patients with SSc and disease models. Levels of A20 are significantly reduced in SSc skin and lungs, while DREAM is elevated. In isolated fibroblasts, A20 mitigates ex vivo profibrotic responses. Mice haploinsufficient for A20, or harboring fibroblasts-specific A20 deletion, recapitulate major pathological features of SSc, whereas DREAM-null mice with elevated A20 expression are protected. In DREAM-null fibroblasts, TGF-β induces the expression of A20, compared to wild-type fibroblasts. An anti-fibrotic small molecule targeting cellular adiponectin receptors stimulates A20 expression in vitro in wild-type but not A20-deficient fibroblasts and in bleomycin-treated mice. Thus, A20 has a novel cell-intrinsic function in restraining fibroblast activation, and together with DREAM, constitutes a critical regulatory network governing the fibrotic process in SSc. A20 and DREAM represent novel druggable targets for fibrosis therapy.