Profibrotic Actions Research Articles

Anti-inflammatory and anti-fibrotic effects of modafinil in nonalcoholic liver disease

Small- and intermediate-conductance Ca2+-activated K+ channels, KCa2.3 and KCa3.1, are involved in cellular signaling processes associated with inflammation and fibrosis. KCa2.3 and KCa3.1 are upregulated by proinflammatory cytokines and profibrotic growth factors. Cyclic AMP, which downregulates KCa2.3 and KCa3.1, is elevated by modafinil in cells; accordingly, we investigated whether modafinil exerts anti-inflammatory and anti-fibrotic responses via KCa2.3- and KCa3.1-mediated pathways in high-fat diet (HFD)- or thioacetamide-induced liver disease models in mice. Modafinil was administered orally in the form of a racemate, (R)-isomer, or (S)-isomer. We also determined whether the treatment targeted the profibrotic activity of hepatic stellate cells using immortalized human hepatic stellate cells (LX-2 cells). Modafinil improved HFD- or thioacetamide-induced changes compared to the control, leading to a reduced inflammatory response, collagen deposition, and α-smooth muscle actin expression both in vivo and in vitro. However, modafinil did not relieve HFD-induced steatosis. There were no significant differences in the effects of the (R)- and (S)-isomers of modafinil. KCa2.3 and KCa3.1 were upregulated and catalase was downregulated in liver tissues from thioacetamide- or HFD-induced liver disease models or in TGF-β-treated LX-2 cells. TGF-β-induced upregulation of KCa2.3, KCa3.1, collagen, and α-smooth muscle actin and downregulation of catalase were reversed by modafinil, polyethylene glycol catalase, N-acetylcysteine, siRNA against KCa2.3 or KCa3.1, and Epac inhibitors. Our investigation revealed that modafinil attenuated inflammatory and fibrotic progression via KCa2.3- and KCa3.1-mediated pathways in nonalcoholic hepatitis, suggesting that inhibiting KCa2.3- and KCa3.1-mediated signaling may serve as a novel therapeutic approach for inflammatory and fibrotic liver diseases.

Dysregulation of Type 1 Interferon Signaling in Systemic Sclerosis: a Promising Therapeutic Target?

There are several lines of evidence at the genetic and gene expression levels linking type I interferon (IFN) activation to systemic sclerosis (SSc) pathogenesis. Herein, we summarize the potential role of type I IFN signaling components as therapeutic targets. All type I IFN cytokines signal through the interferon-α/β receptor (IFNAR). Early phase studies indicate that anifrolumab (a human monoclonal antibody against IFNAR subunit 1) has an acceptable safety profile and can attenuate transforming growth factor beta (TGF-β)-mediated fibrosis in SSc skin, supporting its further clinical development. Janus kinase (JAK) signaling pathways are downstream from IFNAR. Building on their efficacy in hereditary interferonopathies, JAK inhibitors have the potential to block the deleterious IFN and other profibrotic cytokine activation in SSc and are promising drug targets. Moreover, interferon regulator factor (IRF) 5, 7, and 8 have been linked to the profibrotic response in SSc preclinical studies, underscoring their potential as therapeutic targets. Lastly, depletion of plasmacytoid dendritic cells (pDCs) attenuates the IFN activation and fibrotic response in vitro and murine model experiments and can be studied as a viable drug target in future clinical studies. There is increasing evidence linking the prominent type I IFN activation to the observed exaggerated fibrotic response in SSc. Key components of type I IFN signaling are druggable therapeutic targets that can be pursued in future randomized clinical trials, in order to develop more effective therapeutic options for SSc.

Basigin genetic depletion reduces thoracic aortic aneurysm formation in marfan syndrome mice

Abstract Background Marfan Syndrome (MFS) is a rare genetic disease due to mutations in fibrillin 1 gene [1]. It is characterized by thoracic aortic aneurysm (TAA), whose dissection is the major cause of morbidity and mortality in MFS patients [2]. While pathological mechanisms underlying MFS extracellular matrix remodelling have started to be elucidated, an etiological treatment is not available yet [3,4]. Recently, EMMPRIN has been proposed as new actor in pro-fibrotic processes associated with TAA development in MFS patients [4,5]. Purpose To confirm the role of EMMPRIN (i.e. Basigin in mice, Bsg) in TAA formation and progression in a preclinical setting, we assessed whether Bsg genetic depletion in a mouse model of MFS (Fbn1C1039G/+) [6] had an impact on aortic phenotype. Methods We generated a double transgenic mouse model, Fbn1C1039G/+-Bsg+/−, by crossing commercially available Fbn1C1039G/+ with Bsg+/− mice (provided by Dr. Kadomatsu) [7] and we prospectively measured aortic root size in 12 Fbn1C1039G/+-Bsg+/−, 9 Fbn1C1039G/+, and 15 WT mice every 4 weeks from 12 to 20 weeks of age by 2D-echocardiography. Both qRT-PCR and Western Blot were used to evaluate EMMPRIN downstream pro-fibrotic pathway activation in thoracic aortic tissue obtained from mice sacrificed at 12 weeks. Plasma soluble EMMPRIN (sEMMPRIN) levels were assessed by ELISA. Results Echocardiographic assessment at 12 and 20 weeks of age confirmed significantly dilated aortic root in Fbn1C1039G/+ vs WT (mean±SD at 12 weeks: 1.772±0.081 vs 1.394±0.068 mm, respectively, p<0.0001; 20 weeks: 1.899±0.103 vs 1.520±0.083 mm, respectively, p<0.0001), and revealed that Fbn1C1039G/+-Bsg+/− mice had lower aortic root diameter extent compared to Fbn1C1039G/+ (12 weeks: 1.667±0.109 vs 1.772±0.081 mm, respectively, p=0.026; 20 weeks: 1.752±0.129 vs 1.899±0.103 mm, respectively, p=0.011). Pro-fibrotic gene profiling in a subset of 12 weeks old mice showed that Col1A1, Col3A1 and FN1 genes were more expressed in Fbn1C1039G/+ than in WT mice and that the up-regulation of these genes was mitigated in Fbn1C1039G/+ by Bsg depletion (ANOVA p=0.048, p=0.055, and p=0.0003, respectively). Moreover, we found a significant activation of the EMMPRIN downstream pro-fibrotic ERK1/2 pathway in Fbn1C1039G/+ compared to WT mice which was attenuated in the Fbn1C1039G/+-Bsg+/− model (p=0.030). Plasma sEMMPRIN levels were lower in Fbn1C1039G/+ than in WT mice, consistent with what we previously observed in MFS patients [5], and decreased over time in both Fbn1C1039G/+ and WT mice (2-way ANOVA disease p=0.013, time p=0.012, interaction p=ns). Conclusions Basigin genetic depletion reduces dilatation of the aortic root and pro-fibrotic pathway activation in a preclinical MFS model, confirming previous proof-of-concept findings and paving the way for the development of innovative therapies for MFS patients. Plasma sEMMPRIN levels stand as promising tool to predict the aortic enlargement progression in MFS models and patients. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): Italian Ministery of Health

A Catecholaldehyde Metabolite of Norepinephrine Induces Myofibroblast Activation and Toxicity via the Receptor for Advanced Glycation Endproducts: Mitigating Role of l-Carnosine.

Monoamine oxidase (MAO) is rapidly gaining appreciation for its pathophysiologic role in cardiac injury and failure. Oxidative deamination of norepinephrine by MAO generates H2O2 and the catecholaldehyde 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), the latter of which is a highly potent and reactive electrophile that has been linked to cardiotoxicity. However, many questions remain as to whether catecholaldehydes regulate basic physiological processes in the myocardium and the pathways involved. Here, we examined the role of MAO-derived oxidative metabolites in mediating the activation of cardiac fibroblasts in response to norepinephrine. In neonatal murine cardiac fibroblasts, norepinephrine increased reactive oxygen species (ROS), accumulation of catechol-modified protein adducts, expression and secretion of collagens I/III, and other markers of profibrotic activation including STAT3 phosphorylation. These effects were attenuated with MAO inhibitors, the aldehyde-scavenging dipeptide l-carnosine, and FPS-ZM1, an antagonist for the receptor for advanced glycation endproducts (RAGE). Interestingly, treatment of cardiac fibroblasts with a low dose (1 μM) of DOPEGAL-modified albumin phenocopied many of the effects of norepinephrine and also induced an increase in RAGE expression. Higher doses (>10 μM) of DOPEGAL-modified albumin were determined to be toxic to cardiac fibroblasts in a RAGE-dependent manner, which was mitigated by l-carnosine. Collectively, these findings suggest that norepinephrine may influence extracellular matrix remodeling via an adrenergic-independent redox pathway in cardiac fibroblasts involving the MAO-mediated generation of ROS, catecholaldehydes, and RAGE. Furthermore, since elevations in the catecholaminergic tone and oxidative stress in heart disease are linked with cardiac fibrosis, this study illustrates novel drug targets that could potentially mitigate this serious disorder.

Effects of Sex and 17 β-Estradiol on Cardiac Fibroblast Morphology and Signaling Activities In Vitro.

Several studies have demonstrated estrogen’s cardioprotective abilities in decreasing the fibrotic response of cardiac fibroblasts (CFs). However, the majority of these studies are not sex-specific, and those at the cellular level utilize tissue culture plastic, a substrate with a much higher stiffness than physiological conditions. Understanding the intrinsic differences between male and female CFs under more physiologically “healthy” conditions will help to elucidate the divergences in their complex signaling networks. We aimed to do this by conducting a sex-disaggregated analysis of changes in cellular morphology and relative levels of profibrotic signaling proteins in CFs cultured on 8 kPa stiffness plates with and without 17 β-estradiol (E2). Cyclic immunofluorescent analysis indicated that there was a negligible change in cellular morphology due to sex and E2 treatment and that the differences between male and female CFs occur at a biochemical rather than structural level. Several proteins corresponding to profibrotic activity had various sex-specific responses with and without E2 treatment. Single-cell correlation analysis exhibited varied protein–protein interaction across experimental conditions. These findings demonstrate the need for further research into the dimorphisms of male and female CFs to develop better tailored sex-informed prevention and treatment interventions of cardiac fibrosis.

Changes in profibrotic activity in cardiovascular diseases

The myocardium consists of several types of cells: cardiomyocytes, cardiac fibroblasts, endothelial cells and smooth muscle cells. Fibroblasts are cells of mesenchymal origin and are present in all tissues in the body. Cardiac damage can activate available CFBs, provoke transformation of endothelial or epithelial cells into fibroblasts, or induce the production of CFBs from hematopoietic cells and bone marrow. The change in ECM is a key point in the remodeling of the heart in response to the disease process. Disruption of the reticular structure of the ECM alters the connection between myocardial cells and blood vessels, thereby disrupting the structure and function of the heart muscle. Type I and III collagen fibrils are the predominant part of the ECM of the heart. They are synthesized as procollagen, which is converted to a mature collagen molecule. Procollagen type I propeptide (PICP), amino-terminal propeptide type I procollagen (PINP) and N terminal type III collagen peptide (PIIINP) are released in proportional amounts in the synthesis of collagen types I and III and can be used as serum markers for these processes. On the other hand the differentiation from CFBs to myoFB is supported by the transforming growth factor beta (TGF-β), connective tissue growth factor (CTGF), a number of cytokines in the ECM and others. The scientific community is faced with the question of which biomarkers to use to identify the early stages of development of cardiac fibrosis, as well as how to assess the degree of progression of this pathological process.

Changes in profibrotic activity in cardiovascular diseases

Changes in profibrotic activity in cardiovascular diseases

RhoA/ROCK Signaling Regulates TGF-β1-Induced Fibrotic Effects in Human Pterygium Fibroblasts through MRTF-A

ABSTRACT Purpose The overexpression of transforming growth factor-beta1 (TGF-β1) after surgical excision often leads to excessive fibrosis, indicating the recurrence of pterygium. The aims of the present in vitro study were to investigate the role of RhoA/ROCK signaling in regulating fibrotic effects of primary human pterygium fibroblasts (HPFs), as well as to explore the possible mechanisms of these effects. Methods Pterygium samples were obtained from surgery, and profibrotic activation was induced by TGF-β1. Cell proliferation was detected by CCK-8 assay; cell migration was detected by wound healing assay; quantitative real-time PCR and Western blot were used to detect the effects of TGF-β1 and the role of RhoA/ROCK signaling in the synthesis of alpha-smooth muscle actin (a-SMA), type I and III collagen (COL1 and COL3), and matrix metalloproteinase-9 (MMP9) in HPFs. The changes of signaling pathways were detected by Western blot; and pharmaceutical inhibition of RhoA/ROCK signaling and its downstream MRFT-A/SRF transcription pathway were used to assess their possible mechanism in HPFs fibrosis. Results ROCK inhibitor Y-27632 decreased TGF-β1-induced cell proliferation and migration, reduced the TGF-β1-induced expression of profibrotic markers in HPFs, and suppressed TGF-β1-induced nuclear accumulation of Myocardin-related transcription factor A (MRTF-A) as well as accompanied elevation of F/G-actin ratio in HPFs. MRTF-A/Serum response factor (SRF) inhibitor CCG-100602 attenuated the TGF-β1-induced α-SMA expression and reduced myofibroblast activation in HPFs. Conclusions RhoA/ROCK signaling played a pivotal role in TGF-β1-induced fibrosis and myofibroblast activation in HPFs at least in part by inactivating the downstream MRTF-A/SRF transcriptional pathway.

Nintedanib downregulates the transition of cultured systemic sclerosis fibrocytes into myofibroblasts and their pro-fibrotic activity

BackgroundCirculating fibrocytes are an important source of fibroblasts and myofibroblasts, which are involved in fibrotic processes, including systemic sclerosis (SSc).The study aimed to investigate the effect of nintedanib (a tyrosine kinase inhibitor) in inhibiting the in vitro transition of circulating SSc fibrocytes into myofibroblasts and their pro-fibrotic activity.MethodsCirculating fibrocytes were obtained from 18 SSc patients and 5 healthy subjects (HSs). Cultured SSc fibrocytes were maintained in growth medium (untreated cells) or treated with nintedanib 0.1 and 1 μM for 3 and 24 h. Fibroblast-specific protein-1 (S100A4) and α-smooth muscle actin (αSMA), as markers of fibroblast/myofibroblast phenotype, together with type I collagen (COL1) and fibronectin (FN), were investigated by qRT-PCR and Western blotting. Non-parametric tests were used for statistical analysis.ResultsSignificantly elevated gene and protein expressions of αSMA, S100A4, COL1, and FN were observed in SSc fibrocytes compared to HS fibrocytes (gene: αSMA p < 0.001; others p < 0.0001; protein: all p < 0.05). Interestingly, an increased gene and protein expression of αSMA and S100A4 was found in fibrocytes from SSc patients positive for anti-Scl70 and with interstitial lung disease (ILD) (Scl70+ILD+) compared to Scl70−ILD− patients (S100A4: gene: p < 0.01; protein: p < 0.05), whereas no differences were observed for COL1 and FN.Nintedanib reduced gene and protein expression of αSMA, S100A4, COL1, and FN in SSc fibrocytes compared to untreated ones with different statistical significance.Noteworthy, nintedanib significantly downregulated gene and protein expression of αSMA, S100A4, COL1, and FN in Scl70+ILD+ fibrocytes (all p < 0.05), whereas only that of S100A4 and FN was significantly downregulated (p < 0.05) in Scl70−ILD− fibrocytes compared to the related untreated cells.ConclusionsNintedanib seems to downregulate in vitro the transition of fibrocytes into myofibroblasts and their pro-fibrotic activity, particularly in cells isolated from Scl70+ILD+ SSc patients.

TGFβ promotes low IL10-producing ILC2 with profibrotic ability involved in skin fibrosis in systemic sclerosis

ObjectiveInnate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin...

Incorporating mucosal-associated invariant T cells into the pathogenesis of chronic liver disease.

Mucosal-associated invariant T (MAIT) cells have been described in liver and non-liver diseases, and they have been ascribed antimicrobial, immune regulatory, protective, and pathogenic roles. The goals of this review are to describe their biological properties, indicate their involvement in chronic liver disease, and encourage investigations that clarify their actions and therapeutic implications. English abstracts were identified in PubMed by multiple search terms, and bibliographies were developed. MAIT cells are activated by restricted non-peptides of limited diversity and by multiple inflammatory cytokines. Diverse pro-inflammatory, anti-inflammatory, and immune regulatory cytokines are released; infected cells are eliminated; and memory cells emerge. Circulating MAIT cells are hyper-activated, immune exhausted, dysfunctional, and depleted in chronic liver disease. This phenotype lacks disease-specificity, and it does not predict the biological effects. MAIT cells have presumed protective actions in chronic viral hepatitis, alcoholic hepatitis, non-alcoholic fatty liver disease, primary sclerosing cholangitis, and decompensated cirrhosis. They have pathogenic and pro-fibrotic actions in autoimmune hepatitis and mixed actions in primary biliary cholangitis. Local factors in the hepatic microenvironment (cytokines, bile acids, gut-derived bacterial antigens, and metabolic by-products) may modulate their response in individual diseases. Investigational manipulations of function are warranted to establish an association with disease severity and outcome. In conclusion, MAIT cells constitute a disease-nonspecific, immune response to chronic liver inflammation and infection. Their pathological role has been deduced from their deficiencies during active liver disease, and future investigations must clarify this role, link it to outcome, and explore therapeutic interventions.

Renin Angiotensin System Inhibition as treatment for Covid-19?

Renin Angiotensin System Inhibition as treatment for Covid-19?

Calcium Regulation on the Atrial Regional Difference of Collagen Production Activity in Atrial Fibrogenesis.

Background: Atrial fibrosis plays an important role in the genesis of heart failure and atrial fibrillation. The left atrium (LA) exhibits a higher level of fibrosis than the right atrium (RA) in heart failure and atrial arrhythmia. However, the mechanism for the high fibrogenic potential of the LA fibroblasts remains unclear. Calcium (Ca2+) signaling contributes to the pro-fibrotic activities of fibroblasts. This study investigated whether differences in Ca2+ homeostasis contribute to differential fibrogenesis in LA and RA fibroblasts. Methods: Ca2+ imaging, a patch clamp assay and Western blotting were performed in isolated rat LA and RA fibroblasts. Results: The LA fibroblasts exhibited a higher Ca2+ entry and gadolinium-sensitive current compared with the RA fibroblasts. The LA fibroblasts exhibited greater pro-collagen type I, type III, phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), phosphorylated phospholipase C (PLC), stromal interaction molecule 1 (STIM1) and transient receptor potential canonical (TRPC) 3 protein expression compared with RA fibroblasts. In the presence of 1 mmol/L ethylene glycol tetra-acetic acid (EGTA, Ca2+ chelator), the LA fibroblasts had similar pro-collagen type I, type III and phosphorylated CaMKII expression compared with RA fibroblasts. Moreover, in the presence of KN93 (a CaMKII inhibitor, 10 μmol/L), the LA fibroblasts had similar pro-collagen type I and type III compared with RA fibroblasts. Conclusion: The discrepancy of phosphorylated PLC signaling and gadolinium-sensitive Ca2+ channels in LA and RA fibroblasts induces different levels of Ca2+ influx, phosphorylated CaMKII expression and collagen production.

Fibrosis: Sirtuins at the checkpoints of myofibroblast differentiation and profibrotic activity.

Fibrotic diseases are still a serious concern for public health, due to their high prevalence, complex etiology and lack of successful treatments. Fibrosis consists of excessive accumulation of extracellular matrix components. As a result, the structure and function of tissues are impaired, thus potentially leading to organ failure and death in several chronic diseases. Myofibroblasts represent the principal cellular mediators of fibrosis, due to their extracellular matrix producing activity, and originate from different types of precursor cells, such as mesenchymal cells, epithelial cells and fibroblasts. Profibrotic activation of myofibroblasts can be triggered by a variety of mechanisms, including the transforming growth factor-β signalling pathway, which is a major factor driving fibrosis. Interestingly, preclinical and clinical studies showed that fibrotic degeneration can stop and even reverse by using specific antifibrotic treatments. Increasing scientific evidence is being accumulated about the role of sirtuins in modulating the molecular pathways responsible for the onset and development of fibrotic diseases. Sirtuins are NAD+ -dependent protein deacetylases that play a crucial role in several molecular pathways within the cells, many of which at the crossroad between health and disease. In this context, we will report the current knowledge supporting the role of sirtuins in the balance between healthy and diseased myofibroblast activity. In particular, we will address the signalling pathways and the molecular targets that trigger the differentiation and profibrotic activation of myofibroblasts and can be modulated by sirtuins.

FC 102PD INDUCED ARTERIOLAR AND PERITONEAL PATHOMECHANISMS ARE PARTIALLY REVERSED AFTER KIDNEY TRANSPLANTATION

Abstract Background and Aims Due to the unphysiological composition of PD fluids, chronic peritoneal dialysis (PD) induces progressive peritoneal fibrosis, hypervascularization, and vasculopathy. The evolution of the PD membrane and vasculopathy following kidney transplantation (KTx) is largely unknown. Method Arteriolar and peritoneal tissues were obtained from 107 children with chronic kidney disease (CKD5), 72 children on PD (treated with neutral pH PD fluids, with low glucose degradation product content, GDP) and 21 children, who underwent KTx 4-5 weeks after a median 21 months of PD. Specimen underwent standardized digital quantitative histomorphometry. Molecular mechanisms were studied in omental arterioles microdissected from surrounding fat by multi-omics followed by Gene Set Enrichment Analysis (GSEA); key findings were validated in parietal tissues of independent, matched cohorts by quantitative immunohistochemistry (n=15/group). Results Arteriolar transcriptome and proteome GSEA revealed suppression of leucocyte migration and T-cell activation / secretory pathways regulation, of sprouting angiogenesis biological processes and of epithelial proliferation and cell cycle after KTx as compared to PD. Lipid / fatty acid metabolism, autophagy and ATP synthesis pathways were activated. Transcriptome analysis including KTx, PD and CKD5 specifically attributed regulation of arteriolar lipid and fatty acid metabolism to transplantation and comprised 140 transcripts; their regulation was confirmed on the proteome level. Hub gene fatty acid synthase was identified by protein interaction analysis (string-db.org). 15 arteriolar genes activated by PD were inactivated after KTx and included glucose metabolisms and cytoskeleton related transcripts. 24 transcripts and 10 corresponding proteins induced by PD were still active after KTx and associated with biological processes related to TGF-ß signaling, fibrosis and mineral absorption. In line with arteriolar multi-omics findings, peritoneal hypervascularization induced by chronic PD was reversed after Tx to CKD5 level. CD45 positive tissue infiltrating leucocytes count was reduced by 40% and was independently associated with microvessel density in multivariable analysis including PD vintage, daily GDP exposure and recent KTx. Peritoneal lymphatic vessel density, submesothelial thickness, activated fibroblast, fibrin deposit, macrophage and EMT cell counts remained unchanged after KTx compared to PD. Arteriolar lumen to vessel ratios (a marker of vasculopathy) were similar in both groups. Vessel-homeostasis-related proteins in independent, matched cohorts demonstrated increased caspase-3 abundance in peritoneal arterioles after KTx. Arteriolar VEGF-A, thrombospondin, angiopoietin1/2, and hypoxia-inducible factor-1 (HIF-1a) were unchanged, while submesothelial HIF-1a and angiopoietin1/2 were decreased after Tx, favoring vessel maturation. The abundance of the key driver of fibrosis, TGF-ß-effector pSMAD2/3, was unchanged in the peritoneum and arterioles after Tx. Conclusion Our multi-omics analyses of fat covered omental arterioles, not directly exposed to PD fluids, demonstrate inhibition of PD induced immune response and angiogenesis pathways, of glucose metabolism and cytoskeleton regulation to levels similar as seen in children with CKD5. Arteriolar lipid and fatty acid metabolism is selectively altered after KTx. Reversal of low GDP PD induced hypervascularization and inflammation of the parietal peritoneum after KTx, mirror molecular changes in omental arterioles, while profibrotic activity persists after KTx in omental arterioles and in the parietal peritoneum.

The long non-coding RNA PFI protects against pulmonary fibrosis by interacting with splicing regulator SRSF1.

Pulmonary fibrosis (PF) is a type of interstitial pneumonia with complex etiology and high mortality, characterized by progressive scarring of the alveolar interstitium and myofibroblastic lesions. Recently, there has been growing appreciation of the importance of long non-coding RNAs (lncRNAs) in organ fibrosis. The aim of this study was to investigate the role of lncRNAs in lung fibrosis. We used a qRT-PCR assay to identify dysregulated lncRNAs in the lungs of mice with experimental, bleomycin (BLM)-induced pulmonary fibrosis, and a series of molecular assays to assess the role of the novel lncRNA NONMMUT060091, designated as pulmonary fibrosis inhibitor (PFI), which was significantly downregulated in lung fibrosis. Functionally, knockdown of endogenous PFI by smart silencer promoted proliferation, differentiation, and extracellular matrix (ECM) deposition in primary mouse lung fibroblasts (MLFs). In contrast, overexpression of PFI partially abrogated TGF-β1-induced fibrogenesis both in MLFs and in the human fetal lung fibroblast MRC-5 cells. Similarly, PFI overexpression attenuated BLM-induced pulmonary fibrosis compared with wild type (WT) mice. Mechanistically, using chromatin isolation by RNA purification-mass spectrometry (ChIRP-MS) and an RNA pull-down assay, PFI was found to directly bind Serine/arginine-rich splicing factor 1 (SRSF1), and to repress its expression and pro-fibrotic activity. Furthermore, silencing of SRSF1 inhibited TGF-β1-induced proliferation, differentiation, and ECM deposition in MRC-5 cells by limiting the formation of the EDA+Fn1 splicing isoform; whereas forced expression of SRSF1 by intratracheal injection of adeno-associated virus 5 (AAV5) ablated the anti-fibrotic effect of PFI in BLM-treated mice. Overall, these data reveal that PFI mitigated pulmonary fibrosis through negative regulation of the expression and activity of SRSF1 to decrease the formation of the EDA+Fn1 splicing isoform, and suggest that PFI and SRSF1 may serve as potential targets for the treatment of lung fibrosis.

Saikosaponin-d protects against liver fibrosis by regulating the estrogen receptor-β/NLRP3 inflammasome pathway.

Liver fibrosis is the most common pathway in most types of chronic liver damage, characterized by an imbalance of ECM degradation and synthesis. Saikosaponin-d (SSd) possesses anti-inflammatory and anti-fibrotic effects. However, the underlying mechanism by which SSd represses hepatic stellate cell (HSC) activation remains unclear. Here, we found that SSd remarkably alleviated carbon tetrachloride (CCl4)-induced liver fibrosis, as evidenced by decreased collagen levels and profibrotic marker (COl1a1 and α-smooth muscle actin (SMA)) expression. SSd repressed CCl4-induced NOD-like receptor family pyrin-domain-containing-3 (NLRP3) activation in fibrotic livers, as suggested by decreased levels of NLRP3, IL-18, and IL-β. The primary HSCs of CCl4 mice exhibited a significant increase in profibrotic marker expression and NLRP3 activation, but SSd treatment reversed this effect. SSd also repressed TGF-β-induced profibrotic marker expression and NLRP3 activation in vitro. Mechanistically, TGF-β decreased the expression of estrogen receptor-β (ERβ) in HSCs, whereas SSd treatment reversed this effect. ERβ inhibition enhances NLRP3 activation in HSCs. More importantly, ERβ or NLRP3 inhibition partially destroyed the function of SSd in liver fibrosis. In summary, the current data suggest that SSd prevents hepatic fibrosis by regulating the ERβ/NLRP3 inflammasome pathway and suggests SSd as a potential agent for treating liver fibrosis.

NHE1 exacerbates profibrotic activity in idiopathic pulmonary fibrosis and post‐COVID‐19 infection

Idiopathic pulmonary fibrosis (IPF) is a destructive, aging-related pulmonary disease characterized by formation of lung scar tissue. Like other coronaviruses, SARS-COV-2 leads to long term lung damage in some patients in the form of lung fibrosis. In either disease, aggregation of fibroblasts, cell differentiation and excessive tissue remodeling causes the lung tissue to lose lung compliance and oxygen exchange.Sodium hydrogen exchanger isoform 1 (NHE1) is a membrane bound transport protein that functions to support cell movement, regulate cell activity, and balance intracellular pH. A multitude of signaling pathways involved in the regulating of NHE1 function are also involved in the profibrotic activity of fibroblasts and myofibroblasts; therefore we investigated if NHE1 is involved in profibrotic activity and tissue damage associated with IPF and COVID-19 infection. Because NHE1 is key in the remodeling of actin stress fibers and is involved in stiffness of both disorders, we first investigated the impact of NHE1 on stress fiber formation using agonists involved with both fibrosis and COVID response. Lung fibroblasts were treated with three agonists: transforming growth factor-β (TGF-β), serotonin (5HT), and lysophosphatidic acid (LPA) in the presence and absence of an NHE1 inhibitor, ethyl-isopropyl amiloride (EIPA). LPA induced stress fiber formation 65.40+/-3.3 percent over the control, and both 5HT and TGF-β also stimulated the formation of stress fibers by 50.27+/−5.7 and 50.91+/−1.4 percent over the untreated cells. In each case, inhibiting NHE1 with EIPA blocked agonist-activated stress fiber formation, delineating that all three profibrotic factors need NHE1 activity for cytoskeletal remodeling. Another indicator of fibrosis is the fibroblast to myofibroblast transdifferentiation. Myofibroblasts are apoptosis-resistant cells that excessively deposit extracellular matrix proteins; the build up of these cells further compromises the structural integrity of the alveoli and contributes to lung dysfunction. TGF- β, a proinflammatory cytokine, significantly contributes to profibrotic activity by stimulating both fibroblast-myofibroblast transition and excess collagen production. Cells treated with TGF-β to induce cell differentiation showed delayed or significantly reduced myofibroblast transformation in the presence of pharmacological inhibitors of NHE1. Specifically, expression of α-smooth muscle actin (α-SMA), the marker for myofibroblast differentiation, was diminished in each CCL39, LL29, and WI38 cell lines by at least 30 percent and a several day delay of the marker's expression. This study demonstrates the potential for NHE1 as a novel target to fight IPF and lung fibrosis in post-COVID-19 infection.

Cytokine regulation of inflammatory processes in respiratory organs of rats exposed to the combined inhalation of chemicals in low concentrations

Introduction. Morphological studies of animals (trachea, bronchi, lungs) exposed to the combined inhalation of chemicals in low concentrations showed the progression of structural changes, indicating the activation of inflammation and fibrosis in the lungs. The role of cytokine markers in developing inflammatory and fibrotic processes and remodeling lung tissue has been studied. Materials and methods. Male rats (180-200 g) were exposed to a mixture of chemicals (acetone, acetaldehyde, benzene) in low concentrations of 0.7-1.5; 0.9-1.4; 0.2-0.4 (mg/m3), respectively. The concentrations of IL-6, IL-10, IL-1b, IL-4, TGFβ1, TNFα cytokines (pg/ml) have been measured in the lung homogenate by enzyme-linked immunosorbent assay (ELISA). Microscopic anatomy of the lungs, tracheal wall, bronchi has been studied on the 30th day of exposure and the 15th and 90th days of the recovery period. Results. An increase in interleukin-4 and transforming growth factor TGFβ1 in the homogenate of the lung tissue was shown. An increase in lymphatic follicles, the number of lymphocytes, neutrophils, macrophages, and focal accumulations of eosinophils has been observed in the tracheal wall. In lymphoid infiltrates of the lung tissue - eosinophils, macrophages, and plasmocytes. Accumulation of eosinophilic exudate has been observed in some alveoli. The 90th day of the recovery period is characterized by a significant increase of TGFβ1 in the lung tissue, indicating fibrosis, as evidenced by the rise in the number of fibroblasts between the alveoli in the atelectasis zones of lungs. Conclusion. The chronic combined exposure to the mixture of chemicals in low concentrations is accompanied by a pro-inflammatory process in the lungs with the type II hypersensitivity and increasing IL-4 and TGFβ1 (a key mediator of profibrotic activity).

Role of the ROS-JNK Signaling Pathway in Hypoxia-Induced Atrial Fibrotic Responses in HL-1 Cardiomyocytes.

By promoting atrial structural remodeling, atrial hypoxia contributes to the development of the atrial fibrillation substrate. Our study aimed to investigate the modulatory effect of hypoxia on profibrotic activity in cultured HL-1 cardiomyocytes and explore the possible signaling transduction mechanisms of profibrotic activity in vitro. Hypoxia (1% O2) significantly and time-dependently increased the expression of hypoxia-inducible factor (HIF)-1α and fibrotic marker proteins collagen I and III (COL1A and COL3A), transforming growth factor (TGF)-β1 and α-smooth muscle actin (SMA). Western blot or immunohistochemistry analysis showed that hypoxia-induced increase in COL1A and COL3A was significantly attenuated by the addition of SP600125 (a specific c-Jun N-terminal kinase [JNK] inhibitor) or expression of dominant-negative JNK before hypoxia treatment. The inhibition of hypoxia-activated phosphorylation of JNK signal components (JNK, MKK4, nuclear c-Jun and ATF-2) by pre-treatment with SP600125 could suppress hypoxia-stimulated HIF-1α upregulation and fibrotic marker proteins expression. Hypoxia significantly increased reactive oxygen species (ROS) production in cultured HL-1 atrial cells. Pre-treatment with N-acetylcysteine significantly abrogated the expression of nuclear HIF-1α, JNK transduction components and fibrotic marker proteins. Taken together, these findings indicated that the hypoxia-induced atrial profibrotic response occurs mainly via the ROS/JNK pathway, its downstream upregulation of HIF-1α and c-Jun/ATF2 phosphorylation and nuclear translocation to up-regulate the expression of fibrosis-related proteins (COL1A, COL3A, TGF-β1 and α-SMA). Our result suggests that suppression of ROS/JNK signaling pathway is a critical mechanism for developing a novel therapeutic strategy against atrial fibrillation.