Role In Fibrogenesis Research Articles

Anti-fibrotic Potential of WJ-MSC Exosomes in Liver Fibrosis: Mechanistic Insights and Dose-Response Efficacy

Background: Hepatic fibrosis is a biological response characterized by the accumulation of extracellular matrix (ECM) during the wound healing process. Hepatic stellate cells (HSCs) play a pivotal role in fibrogenesis, transitioning from quiescent to myofibroblast cell types and leading to excessive ECM production. Platelet-derived growth factor (PDGF), a potent mitogen, is produced by activated HSCs, stimulating cell proliferation and migration. Objectives: This study aims to analyze the impact of Wharton's jelly mesenchymal stem cell (WJ-MSC)-derived exosomes on HSC activation induced by PDGF during liver fibrosis. Methods: Hepatic stellate cells-T6 cells were treated with PDGF-BB for 24 hours to induce activation, followed by treatment with varying concentrations of WJ-MSC-derived exosomes (0, 25, and 50 µg/mL) for another 24 hours. The effects of exosome treatment on HSC activation were evaluated through flow cytometry, differentiation assays, dynamic light scattering (DLS), transmission electron microscopy (TEM), RT-PCR, and western blot analysis. Results: Our study yields promising results, highlighting the potential therapeutic effects of WJ-MSC-derived exosomes on liver fibrosis. The dose-dependent decrease in fibrotic markers such as α-SMA, COLA1, and phosphorylated AKT protein in PDGF-BB-treated HSC-T6 cells suggests that WJ-MSC exosomes exert an anti-fibrotic effect by inhibiting HSC activation. Conclusions: These findings suggest that exosomes derived from WJ-MSCs hold therapeutic promise for liver fibrosis treatment by targeting key pathways involved in HSC activation and fibrogenesis. Further investigation into the underlying mechanisms of this anti-fibrotic effect and the potential clinical applications of WJ-MSC-derived exosomes in liver fibrosis management is warranted.

The Complex Interplay of TGF-β and Notch Signaling in the Pathogenesis of Fibrosis

Fibrosis is a major medical challenge, as it leads to irreversible tissue remodeling and organ dysfunction. Its progression contributes significantly to morbidity and mortality worldwide, with limited therapeutic options available. Extensive research on the molecular mechanisms of fibrosis has revealed numerous factors and signaling pathways involved. However, the interactions between these pathways remain unclear. A comprehensive understanding of the entire signaling network that drives fibrosis is still missing. The TGF-β and Notch signaling pathways play a key role in fibrogenesis, and this review focuses on their functional interplay and molecular mechanisms. Studies have shown synergy between TGF-β and Notch cascades in fibrosis, but antagonistic interactions can also occur, especially in cardiac fibrosis. The molecular mechanisms of these interactions vary depending on the cell context. Understanding these complex and context-dependent interactions is crucial for developing effective strategies for treating fibrosis.

An L-type calcium channel blocker nimodipine exerts anti-fibrotic effects by attenuating TGF-β1 induced calcium response in an in vitro model of thyroid eye disease

BackgroundThyroid eye disease (TED) is a vision-threatening autoimmune disorder. Orbital tissue fibrosis leading to intractable complications remains a troublesome issue in TED management. Exploration of novel therapeutic targets and agents to ameliorate tissue fibrosis is crucial for TED. Recent work suggests that Ca2+ signaling participates in tissue fibrosis. However, whether an alteration of Ca2+ signaling has a role in fibrogenesis during TED remains unclear. In this study, we aimed to investigate the role of Ca2+ signaling in the fibrogenesis process during TED and the potential therapeutic effects of a highly selective inhibitor of the L-type calcium channel (LTCC), nimodipine, through a TGF-β1 induced in vitro TED model.MethodsPrimary culture of orbital fibroblasts (OFs) were established from orbital adipose connective tissues of patients with TED and healthy control donors. Real-time quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing were used to assess the genes expression associated with LTCC in OFs. Flow cytometry, RT-qPCR, 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay, wound healing assay and Western blot (WB) were used to assess the intracellular Ca2+ response on TGF-β1 stimulation, and to evaluate the potential therapeutic effects of nimodipine in the TGF-β1 induced in vitro TED model. The roles of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 1 (STAT1) in fibrogenesis during TED were determined by immunohistochemistry, WB, flow cytometry and co-immunoprecipitation assay. Selective inhibitors were used to explore the downstream signaling pathways.ResultsLTCC inhibitor nimodipine blocked the TGF-β1 induced intracellular Ca2+ response and further reduced the expression of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (Col1A1) and collagen type I alpha 2 (Col1A2) in OFs. Besides, nimodipine inhibited cell proliferation and migration of OFs. Moreover, our results provided evidence that activation of the CaMKII/STAT1 signaling pathway was involved in fibrogenesis during TED, and nimodipine inhibited the pro-fibrotic functions of OFs by down-regulating the CaMKII/STAT1 signaling pathway.ConclusionsTGF-β1 induces an LTCC-mediated Ca2+ response, followed by activation of CaMKII/STAT1 signaling pathway, which promotes the pro-fibrotic functions of OFs and participates in fibrogenesis during TED. Nimodipine exerts potent anti-fibrotic benefits in vitro by suppressing the CaMKII/STAT1 signaling pathway. Our work deepens our understanding of the fibrogenesis process during TED and provides potential therapeutic targets and alternative candidate for TED.

Fibroblast activation protein and disease severity, progression, and survival in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibrosis in the lungs. Activated fibroblasts play a central role in fibrogenesis and express fibroblast activation protein α. A truncated, soluble form (sFAP) can be measured in blood and is a potential novel biomarker of disease activity. The aim was to study the association between sFAP and clinical, radiological, and histopathological measures of disease severity, progression, and survival in a prospective, multicentre, real-world cohort of patients with IPF. Patients with IPF were recruited from the tertiary interstitial lung disease centres in Denmark and followed for up to 3 years. Baseline serum levels of sFAP were measured by ELISA in patients with IPF and compared to healthy controls. Pulmonary function tests, 6-minute walk test and quality of life measures were performed at baseline and during follow-up. The study included 149 patients with IPF. Median sFAP in IPF was 49.6 ng/mL (IQR: 43.1-61.6 ng/mL) and in healthy controls 73.8 ng/mL (IQR: 62.1-92.0 ng/mL). Continuous sFAP was not associated with disease severity, progression or survival (p > 0.05). After dichotomization of sFAP below or above mean sFAP + 2 SD for healthy controls, higher levels of sFAP were associated with lower FVC % predicted during follow-up (p < 0.01). Higher than normal serum levels of sFAP were associated with longitudinal changes in FVC % predicted, but sFAP did not show clear associations with other baseline or longitudinal parameters. As such, sFAP has limited use as a biomarker of disease progression or survival in patients with IPF.

Mesenchymal stem cells-derived exosomes alleviate liver fibrosis by targeting Hedgehog/SMO signaling.

Despite increasing knowledge regarding the cellular and molecular mechanisms of liver fibrogenesis, there is currently no approved drug for the treatment of liver fibrosis. Mesenchymal stem cells (MSCs) are multipotent progenitor cells representing an attractive therapeutic tool for tissue damage and inflammation. This study was designed to determine the protective effect and underlying mechanism of human umbilical cord-derived MSCs (UC-MSCs) on thioacetamide-induced liver fibrosis. Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide (TAA). Some mice were then given injection of UC-MSCs or UC-MSCs-derived exosomes (UC-MSCs-Exo) via the tail vein. Liver tissues were collected for histologic analysis. We found that administration of UC-MSCs significantly reduced serum alanine aminotransferase and aspartate aminotransferase levels, and attenuated hepatic inflammation and fibrosis. Moreover, the therapeutic effect of UC-MSCs-derived exosomes was similar to that of UC-MSCs. Intriguingly, UC-MSCs-Exo treatment downregulated the expression of smoothened (SMO), a fundamental component of Hedgehog signaling which plays a critical role in fibrogenesis, and subsequently inhibited the activation of hepatic stellate cells, a central driver of fibrosis in experimental and human liver injury. Furthermore, the anti-inflammatory and anti-fibrotic effects of UCMSCs- Exo was reversed by the SMO agonist SAG treatment in mice. Our findings suggest that UC-MSCs-Exo exert therapeutic effects on liver fibrosis, at least in part, through inhibiting the Hedgehog/SMO signaling pathway.

Abstract We108: Adipocyte Enhancer Binding Protein 1 (AEBP1) Inhibition as a Potential Anti-Fibrotic Therapy in Heart Failure

Introduction: Cardiac fibrosis is one of the major hallmarks of heart failure (HF) progression. Fibrosis is initiated as a repair mechanism following any kind of cardiac injury including a heart attack, inflammation, hypertension, etc. Briefly, fibroblasts are activated to form myofibroblasts that secrete collagen. Persistent fibroblast activation leads to excess collagen deposition leading to tissue stiffening and impaired contractility and thereby HF. To date, there is no anti-fibrotic therapy used in HF patients. Using bulk and snRNA sequencing data performed on myocardial tissue from HF patients, we identified adipocyte enhancer binding protein (AEBP1) to be significantly upregulated in activated fibroblasts. AEBP1 has been shown to play a crucial role in fibroblast activation following injury in the kidney, liver, and lungs. AEBP1 inhibition resulted in fibrosis attenuation in these organs. However, its role in cardiac fibrosis is not well understood. Hypothesis: We hypothesize that AEBP1 is crucial for cardiac fibroblast activation and AEBP1 inhibition will prevent collagen secretion and lead to improved cardiac function. Methods: Human cardiac fibroblasts (HCF) from non-failing donors were used for in vitro experiments. AdV-CMV-AEBP1 was used for AEBP1 overexpression (OE) and AdV-CMV-shAEBP1 was used to knockdown (KD) AEBP1 in vitro. AAV9-CMV-shAEBP1 was used for in vivo KD of AEBP1 in mice following myocardial infarction (MI). Human myocardial slices from n=6 HF patients were used and AdV-CMV-shAEBP1 was used to knockdown AEBP1 in human samples. Results and Discussion: AEBP1 OE in HCF resulted in fibroblast activation and collagen secretion (n=6, p=0.002). Contrarily, loss of AEBP1 significantly prevented fibroblast activation, evident from a reduction in transgelin (SM22) (n=6, p=0.02) and collagen (COL1A1) protein levels (n=6, p=0.01). AEBP1 KD in a mouse model of acute cardiac fibrosis resulted in reduced fibrosis and improved cardiac function (n=6, 12% absolute improvement compared to untreated mice, relative improvement in ejection fraction, p=0.02). Tissue culture studies of human HF myocardium showed a significant improvement in cardiac structure evident from reduced cardiomyocyte hypertrophy (n=6 HF patients, p=0.05) and reduced fibrosis (n=6, p&lt;0.0001) following AEBP1 KD. Overall, we show that AEBP1 plays a critical role in fibrogenesis, and its inhibition has the potential to attenuate fibrosis in acute and chronic HF.

Chronic liver disease and fibrosis: A review of emerging biomarkers and therapeutic targets

Chronic liver disease (CLD) and fibrosis represent a significant global health burden, driven by a range of etiologies including viral hepatitis, alcohol abuse, and non-alcoholic fatty liver disease (NAFLD). These conditions often progress to cirrhosis, liver failure, and hepatocellular carcinoma (HCC), underscoring the urgent need for effective diagnostic and therapeutic strategies. Emerging biomarkers and therapeutic targets offer promising avenues for early diagnosis and intervention in CLD and fibrosis. Biomarkers are crucial for the early detection and monitoring of CLD and fibrosis, allowing for timely therapeutic intervention. Serum biomarkers such as liver enzymes (ALT, AST), bilirubin, and platelet count have traditionally been used, but they lack specificity and sensitivity. Recent advances have identified novel biomarkers with improved diagnostic performance. For instance, serum levels of fibrosis markers like hyaluronic acid, procollagen type III N-terminal peptide (P3NP), and tissue inhibitor of metalloproteinases-1 (TIMP-1) have shown potential in assessing liver fibrosis. Additionally, non-invasive imaging techniques such as transient elastography and magnetic resonance elastography provide quantitative measures of liver stiffness, correlating with fibrosis stage. The pathogenesis of liver fibrosis involves complex interactions between hepatocytes, hepatic stellate cells (HSCs), and the extracellular matrix (ECM). HSCs play a central role in fibrogenesis by transforming into myofibroblasts that secrete collagen and other ECM components. Targeting the activation and proliferation of HSCs has emerged as a promising therapeutic strategy. Small molecule inhibitors, such as those targeting the PDGF and TGF-? signaling pathways, have shown efficacy in preclinical models. Furthermore, antifibrotic agents like simtuzumab, an anti-LOXL2 monoclonal antibody, are being evaluated in clinical trials for their potential to halt or reverse fibrosis progression. Another promising approach involves the modulation of the gut-liver axis. Dysbiosis and increased intestinal permeability contribute to liver inflammation and fibrosis. Probiotics, prebiotics, and fecal microbiota transplantation (FMT) are being explored for their potential to restore gut homeostasis and mitigate liver injury. Additionally, the role of the immune system in fibrosis has gained attention, with immune checkpoint inhibitors and anti-inflammatory agents being investigated for their ability to modulate immune responses and reduce fibrosis. Keywords: Chronic Liver Disease, Fibrosis, Biomarkers, Therapeutic Targets.

#2394 Inhibition of ACSS2-mediated H3K9 crotonylation alleviates kidney fibrosis via IL-1β-dependent macrophage activation and tubular cell senescence

Abstract Background and Aims Histone lysine crotonylation (Kcr), a novel posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. Method Human renal tissue, fixed in formaldehyde, and embedded in paraffin, was selected from the files of the Service of Pathology of West China Hospital, Sichuan University.ACSS2 knockout mice and tubular specific knock out of ACSS2 mice in C57BL/6N background and wild-type littermates (WT) were used to induce the FAN and UUO model or as control groups (n = 6 each). In the parallel study, C57BL/6N mice were randomly divided into three groups: control, FAN/UUO and FAN/UUO+ACSS2 inhibitor (n = 6 each). At day 7, mice were sacrificed and the serum creatinine, BUN and urinary albumin/creatinine ratio (ACR) were tested. Renal histological injury was measured by HE and Masson's trichrome staining. SA-β-gal staining was used to check cellular senescence. Renal tissues of the mice were analyzed by western blot and qPCR assay for the expression of ACSS2, H3K9cr, fibrosis and cellular senescence related proteins and genes. RNA-sequencing and ChIP-sequencing combined with in vitro cell experiments were used to figure out the underlying mechanism. Results In this study, we reported that histone Kcr of tubular epithelial cells was abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) was found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys revealed that the hub proinflammatory cytokine IL-1β, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppressed H3K9cr-mediated IL-1β expression, which thereby alleviated IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Conclusion Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

Inhibition of ACSS2-mediated histone crotonylation alleviates kidney fibrosis via IL-1β-dependent macrophage activation and tubular cell senescence

Histone lysine crotonylation (Kcr), as a posttranslational modification, is widespread as acetylation (Kac); however, its roles are largely unknown in kidney fibrosis. In this study, we report that histone Kcr of tubular epithelial cells is abnormally elevated in fibrotic kidneys. By screening these crotonylated/acetylated factors, a crotonyl-CoA-producing enzyme ACSS2 (acyl-CoA synthetase short chain family member 2) is found to remarkably increase histone 3 lysine 9 crotonylation (H3K9cr) level without influencing H3K9ac in kidneys and tubular epithelial cells. The integrated analysis of ChIP-seq and RNA-seq of fibrotic kidneys reveal that the hub proinflammatory cytokine IL-1β, which is regulated by H3K9cr, play crucial roles in fibrogenesis. Furthermore, genetic and pharmacologic inhibition of ACSS2 both suppress H3K9cr-mediated IL-1β expression, which thereby alleviate IL-1β-dependent macrophage activation and tubular cell senescence to delay renal fibrosis. Collectively, our findings uncover that H3K9cr exerts a critical, previously unrecognized role in kidney fibrosis, where ACSS2 represents an attractive drug target to slow fibrotic kidney disease progression.

The Role of Fibrogenesis and Extracellular Matrix Proteins in the Pathogenesis of Graves' Ophthalmopathy.

Graves' ophthalmopathy (GO), or thyroid eye disease (TED), is the most frequent extrathyroidal manifestation of Graves' disease (GD). Inflammation and subsequent aberrant tissue remodeling with fibrosis are important pathogenesis. There are many proposed mechanisms and molecular pathways contributing to tissue remodeling and fibrosis in GO, including adipogenesis, fibroblast proliferation and myofibroblasts differentiation, oxidative stress, endoplasmic reticulum (ER) stress, hyaluronan (HA) and glycosaminoglycans (GAGs) accumulation in the extracellular matrix (ECM) and new concepts of epigenetics modification, such as histone modification, DNA methylation, non-coding RNAs, and gut microbiome. This review summarizes the current understanding of ECM proteins and associated tissue remodeling in the pathogenesis and potential mediators for the treatment of GO.

Pro-Fibrotic Effects of CCL18 on Human Lung Fibroblasts Are Mediated via CCR6.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease of unknown origin, with a median patient survival time of ~3 years after diagnosis without anti-fibrotic therapy. It is characterized by progressive fibrosis indicated by increased collagen deposition and high numbers of fibroblasts in the lung. It has been demonstrated that CCL18 induces collagen and αSMA synthesis in fibroblasts. We aimed to identify the CCL18 receptor responsible for its pro-fibrotic activities. We used a random phage display library to screen for potential CCL18-binding peptides, demonstrated its expression in human lungs and fibroblast lines by PCR and immunostaining and verified its function in cell lines. We identified CCR6 (CD196) as a CCL18 receptor and found its expression in fibrotic lung tissue and lung fibroblast lines derived from fibrotic lungs, but it was almost absent in control lines and tissue. CCL18 induced receptor internalization in a CCR6-overexpressing cell line. CCR6 blockade in primary human lung fibroblasts reduced CCL18-induced FGF2 release as well as collagen-1 and αSMA expression. Knockdown of CCR6 in a mouse fibroblast cell line abolished the induction of collagen and α-smooth muscle actin expression. Our data indicate that CCL18 triggers pro-fibrotic processes via CCR6, highlighting its role in fibrogenesis.

MUC-16, tumour and congestive heart failure marker, is expressed on epicardial mesothelial cells and is inversely associated with epicardial fat genesis

Abstract Background CA-125 or mucin-16 (MUC-16) is a protector protein on epithelial luminal surfaces against physical stress through hydration process. However, some evidence suggests a role in fibrogenesis. MUC-16 can be detected in plasma after proteolytic cleavage with a predictive value for heart failure progression. Our main objective was to study the expression levels of this glycoprotein on epicardial mesothelial cells, its association with epicardial fat genesis, and biochemical markers. Methods and results We have used epicardial and subcutaneous fat from patients(n = 29) undergoing open heart surgery under informed consent and after approval by Ethics Committee. MUC-16 expression levels were detected on formalin-fixed paraffin-embedded epicardial fat biopsies with CA-125 antibody or real-time PCR on stored biopsies (n = 18) at -80ºC. Epicardial cells scraped with a scalpel were cultured and their MUC-16 expression was confirmed with a specific antibody and secondary fluorescent antibody. Primary cultures from epicardial stromal cells of 8 patients were obtained with collagenase digestion. After 15 days, adipogenesis was induced for 15 days with an adipogenic cocktail. Adipogenesis (AdipoQ) and mesothelial (ITLN-1) markers were quantified by real-time PCR on cells with and without adipogenesis. CA-125 expression levels were analyzed by real time-PCR on epicardial fat from 18 patients, as well as inflammation, fibrosis, stretch and adiposity markers levels on plasma by multiplex assay. Our results showed that MUC-16 is localized on epicardial mesothelial cells but not on adipocytes. The MUC-16-stained cells can be cultured and isolated after scraping the epicardium. The isolated epicardial stromal cells with collagenase showed a positive relationship between mesothelial maker (ITLN-1) and CA125 (Figure below). The higher presence of MUC-16 levels was inversely associated with AdipoQ after adipogenesis induction. From 8 plasma studied proteins, thrombospondin-2 (TP-2), was the best associated protein with MUC-16 on epicardial fat (r = 0,472; p = 0,48). Conclusion Epicardial cells express MUC-16. These types of cells can be isolated and cultured for studying their cleavage regulation and modulation. Epicardial stromal cells with a higher percentage of MUC-16 reduce the epicardial fat genesis. Further studies are needed for finding the main circulating plasma levels associated with epicardial MUC-16.

In Vitro Effect of Propofol on the Expression of Genes Involved in Inflammation and Apoptosis in Corneal Activated Keratocytes.

We investigated the effect of propofol (0.5, 5, and 50 μM) on the gene expression of inflammatory cytokines [ IL-1β , IL-6 , transforming growth factor β ( TGF-β ), and LIF ] and apoptosis process ( BCL-2 and Bax ) in corneal activated keratocytes (CAKs). CAKs (10 6 cells/10 cm 2 ) were exposed to propofol at a concentration of 0.5, 5, and 50 μM for 24 hours at 37°C. The control group did not receive propofol at the same time or under the same condition. Ribonucleic acid (RNA) extraction, complementary DNA (cDNA) synthesis, and real-time polymerase chain reaction (PCR) were performed to quantify the relative expression of IL-1β , IL-6 , TGF-β , LIF , BCL-2 , and Bax expression in the treated versus control cells. The results of this study showed that propofol treatment (0.5 and 5 μM) led to the downregulation of IL-1β and IL-6 gene expression in CAKs. TGF-β (with a role in fibrogenesis) was not changed in 0.5 and 5 μM propofol-treated CAKs, whereas CAKs treated with 50 μM propofol showed upregulation of the TGF-β gene. LIF (with a role in regeneration) was upregulated in 0.5 and 5 μM propofol-treated CAKs. The BCL-2/Bax ratio (as the antiapoptosis index) was increased in CAKs treated with 0.5 μM propofol and indicated the induction of an antiapoptotic effect. We showed that CAKs treatment with propofol, at concentrations of 0.5 and 5 μM, could decrease the expression of genes related to inflammation and enhance the genes associated with cell regeneration. While 50 μM propofol treatment might induce CAK fibrogenesis. This proof-of-concept study could preserve a groundwork for future drug design for the treatment of corneal stromal diseases and ocular regenerative medicine.

TGF-β1 stimulation and VDR-dependent activation modulate calcitriol action on skeletal muscle fibroblasts and Smad signalling-associated fibrogenesis

Fibroblasts play a pivotal role in fibrogenesis after skeletal muscle injury. Excess fibrous formation can disrupt contractile functions and delay functional recovery. Although vitamin D receptor (VDR) is expressed explicitly in regenerating muscle compared with uninjured muscle, how calcitriol [1α,25(OH)2D3] directly regulates skeletal muscle primary fibroblast proliferation, the transition to myofibroblasts, and Smad signalling-associated fibrogenesis is currently unknown. Herein, the effects of calcitriol on cultured skeletal muscle primary fibroblasts of male C57BL/6 mice (aged 1 month old) were investigated. The percentage of BrdU+ nuclei in primary fibroblasts was significantly decreased after calcitriol treatment; however, the antiproliferative effect of calcitriol was diminished after TGF-β1 stimulation to induce fibroblast to myofibroblast transition. This suppressive effect was associated with significantly decreased VDR expression in TGF-β1-treated cells. In addition, Vdr siRNA transfection abolished the effects of calcitriol on the suppression of α-SMA expression and Smad2/3 signalling in myofibroblasts, supporting that its antifibrogenic effect requires VDR activation. Compared with calcitriol, the antifibrotic agent suramin could inhibit fibroblast/myofibroblast proliferation and suppress the expression of TCF-4, which regulates fibrogenic determination. Collectively, these findings suggest that profibrotic stimulation and VDR-dependent activation could modulate the effects of calcitriol on skeletal muscle fibroblast proliferation and fibrogenesis processes. Therefore, TGF-β1 and VDR expression levels are crucial determinants for the antifibrogenic effect of calcitriol on skeletal muscle after injury.

Revealing immune infiltrate characteristics and potential immune-related genes in hepatic fibrosis: based on bioinformatics, transcriptomics and q-PCR experiments.

The occurrence and progression of hepatic fibrosis (HF) is accompanied by inflammatory damage. Immune genes play a pivotal role in fibrogenesis and inflammatory damage in HF by regulating immune cell infiltration. However, the immune mechanisms of HF are inadequately studied. Therefore, this research aims to identify the immune genes and biological pathway which involved in fibrosis formation and inflammatory damage in HF and explore immune target-based therapeutics for HF. The expression dataset GSE84044 of HF was downloaded from the GEO database. The crucial module genes for HF were screened according to weighted gene co-expression network analysis (WGCNA). The crucial module genes were mapped to immune-related genes obtained from the ImmPort database to obtain the hepatic fibrosis immune genes (HFIGs). In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed on HFIGs. Then, the protein-protein interaction (PPI) network was conducted on HFIGs and hub genes were identified from the PPI network. Moreover, immune infiltration analysis was performed to identified correlation between hub gene and immune cell infiltration. To verify the reliability of the GSE84044 expression profile data analysis, a rat model of CCl4-induced HF was established, followed by transcriptome sequencing and immunofluorescence analysis and quantitative reverse transcription (q-PCR) experiments were performed in HF rats and normal rat liver tissues. Finally, CMAP platform was used to explore immune target-based therapeutics for HF. In the bioinformatics analysis of GSE84044 data, 98 HFIGs were screened. These genes were mainly involved in inflammation-related biological pathways such as NOD-like receptor signaling pathway, NF-kappa B signaling pathway, Toll-like receptor signaling pathway and PI3K-Akt signaling pathway. From the PPI network, 10 hub genes were identified, including CXCL8, IL18, CXCL10, CD8A, IL7, PTPRC, CCL5, IL7R, CXCL9 and CCL2. Immune infiltration analysis showed that immune cells like neutrophils, natural killer (NK) cells, macrophages M1 and macrophages M2 were significantly correlated with the hepatic fibrosis process and hub gene expression was significantly correlated with these immune cells. Notably, most of the biological pathways HFIGs riched and all the hub gene expression except CXCL8 were validated in subsequent transcriptome and qRCR experiments. Finally, 15 small molecule compounds with the potential to reverse the high expression of hub genes were screen out as potential therapeutic agents for HF. The immune genes CXCL8, IL18, CXCL10, CD8A, IL7, PTPRC, CCL5, IL7R, CXCL9 and CCL2 may play an essential role in the fibrosis formation and inflammatory damage in HF. The outcomes of this research provide a basis for the study of the immune mechanisms of HF and contribute to the diagnosis and prevention and treatment of HF in clinical practice.

Role of Nox4 in Mitigating Inflammation and Fibrosis in Dextran Sulfate Sodium–Induced Colitis

Fibrosis development in ulcerative colitis is associated directly with the severity of mucosal inflammation, which increases the risk of colorectal cancer. The transforming growth factor-β (TGF-β) signaling pathway is an important source of tissue fibrogenesis, which is stimulated directly by reactive oxygen species produced from nicotinamide adenine dinucleotide phosphate oxidases (NOX). Among members of the NOX family, NOX4 expression is up-regulated in patients with fibrostenotic Crohn's disease (CD) and in dextran sulfate sodium (DSS)-induced murine colitis. The aim of this study was to determine whether NOX4 plays a role in fibrogenesis during inflammation in the colon using a mouse model. Acute and recovery models of colonic inflammation were performed by DSS administration to newly generated Nox4-/- mice. Pathologic analysis of colon tissues was performed, including detection of immune cells, proliferation, and fibrotic and inflammatory markers. RNA sequencing was performed to detect differentially expressed genes between Nox4-/- and wild-type mice in both the untreated and DSS-treated conditions, followed by functional enrichment analysis to explore the molecular mechanisms contributing to pathologic differences during DSS-induced colitis and after recovery. Nox4-/- mice showed increased endogenous TGF-β signaling in the colon, increased reactive oxygen species levels, intensive inflammation, and an increased fibrotic region after DSS treatment compared with wild-type mice. Bulk RNA sequencing confirmed involvement of canonical TGF-β signaling in fibrogenesis of the DSS-induced colitis model. Up-regulation of TGF-β signaling affects collagen activation and T-cell lineage commitment, increasing the susceptibility for inflammation. Nox4 protects against injury and plays a crucial role in fibrogenesis in DSS-induced colitis through canonical TGF-β signaling regulation, highlighting a new treatment target.

The Genetic and Epigenetic Footprint in Idiopathic Pulmonary Fibrosis and Familial Pulmonary Fibrosis: A State-of-the-Art Review.

Idiopathic pulmonary fibrosis (IPF) is a rare disease of the lung with a largely unknown etiology and a poor prognosis. Intriguingly, forms of familial pulmonary fibrosis (FPF) have long been known and linked to specific genetic mutations. There is little evidence of the possible role of genetics in the etiology of sporadic IPF. We carried out a non-systematic, narrative literature review aimed at describing the main known genetic and epigenetic mechanisms that are involved in the pathogenesis and prognosis of IPF and FPF. In this review, we highlighted the mutations in classical genes associated with FPF, including those encoding for telomerases (TERT, TERC, PARN, RTEL1), which are also found in about 10-20% of cases of sporadic IPF. In addition to the Mendelian forms, mutations in the genes encoding for the surfactant proteins (SFTPC, SFTPA1, SFTPA2, ABCA3) and polymorphisms of genes for the mucin MUC5B and the Toll-interacting protein TOLLIP are other pathways favoring the fibrogenesis that have been thoroughly explored. Moreover, great attention has been paid to the main epigenetic alterations (DNA methylation, histone modification and non-coding RNA gene silencing) that are emerging to play a role in fibrogenesis. Finally, a gaze on the shared mechanisms between cancer and fibrogenesis, and future perspectives on the genetics of pulmonary fibrosis have been analyzed.

The novel subclusters based on cancer-associated fibroblast for pancreatic adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) is a fatal disease characterized by promoting connective tissue proliferation in the stroma. Activated cancer-associated fibroblasts (CAFs) play a key role in fibrogenesis in PAAD. CAF-based tumor typing of PAAD has not been explored. We extracted single-cell sequence transcriptomic data from GSE154778 and CRA001160 datasets from Gene Expression Omnibus or Tumor Immune Single-cell Hub to collect CAFs in PAAD. On the basis of Seurat packages and new algorithms in machine learning, CAF-related subtypes and their top genes for PAAD were analyzed and visualized. We used CellChat package to perform cell-cell communication analysis. In addition, we carried out functional enrichment analysis based on clusterProfiler package. Finally, we explored the prognostic and immunotherapeutic value of these CAF-related subtypes for PAAD. CAFs were divided into five new subclusters (CAF-C0, CAF-C1, CAF-C2, CAF-C3, and CAF-C4) based on their marker genes. The five CAF subclusters exhibited distinct signaling patterns, immune status, metabolism features, and enrichment pathways and validated in the pan-cancer datasets. In addition, we found that both CAF-C2 and CAF-C4 subgroups were negatively correlated with prognosis. With their top genes of each subclusters, the sub-CAF2 had significantly relations to immunotherapy response in the patients with pan-cancer and immunotherapy. We explored the heterogeneity of five subclusters based on CAF in signaling patterns, immune status, metabolism features, enrichment pathways, and prognosis for PAAD.

Discovery of novel immunotherapeutic drug candidates for sciatic nerve injury using bioinformatic analysis and experimental verification.

Inflammation following nerve injury and surgery often causes peripheral nerve adhesion (PNA) to the surrounding tissue. Numerous investigations independently examined the prevention or inhibition of PNA, however, an intervention targeting macrophages has not been fully elucidated. Basement membrane (BM) genes are known to modulate central nervous system (CNS) inflammation, however, their activities in the peripheral nervous system (PNS) remains undiscovered. In this report, we carried out weighted correlation network analysis (WCNA) to screen for principal sciatic nerve injury (SNI) module genes. Once an association between the module and BM genes was established, the protein-protein interaction (PPI) and immune infiltration analyses were employed to screen for relevant BM-related immune genes (Itgam, SDC1, Egflam, and CD44) in SNI. Subsequently, using the Drug SIGnatures (DSigDB) database and molecular docking, we demonstrated that Trichostatin A (TSA) interacted with key immune genes. TSA is known to enhance M2 macrophage expression and attenuate fibrosis. Nevertheless, the significance of the epigenetic modulation of macrophage phenotypes in dorsal root ganglion (DRG) is undetermined after SNI. In this article, we examined the TSA role in fibrogenesis and macrophage plasticity associated with DRG. We revealed that TSA enhanced M2 macrophage aggregation, inhibited fibroblast activation, and improved sciatic nerve regeneration (SNR) and sensory functional recovery (FR) after SNI. In addition, TSA suppressed M1 macrophages and enhanced M2 macrophage invasion within the DRG tissue. Furthermore, TSA dramatically reduced IL-1β and TNFα levels, while upregulating IL-10 level. In summary, this research revealed for the first time that TSA alleviates fibrosis in DRG by promoting an M1 to M2 macrophage transition, which, in turn, accelerates SNR.

Tracking matricellular protein SPARC in extracellular vesicles as a non-destructive method to evaluate lipid-based antifibrotic treatments

Uncovering the complex cellular mechanisms underlying hepatic fibrogenesis could expedite the development of effective treatments and noninvasive diagnosis for liver fibrosis. The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers as potential alternative to liver biopsies. We developed a solid set of methods to isolate and characterize EVs from differently treated human hepatic stellate cell (HSC) line LX-2, and we investigated their biological effect onto naïve LX-2, proving that EVs do play an active role in fibrogenesis. We mined our proteomic data for EV-associated proteins whose expression correlated with HSC treatment, choosing the matricellular protein SPARC as proof-of-concept for the feasibility of fluorescence nanoparticle-tracking analysis to determine an EV-based HSCs’ fibrogenic phenotype. We thus used EVs to directly evaluate the efficacy of treatment with S80, a polyenylphosphatidylcholines-rich lipid, finding that S80 reduces the relative presence of SPARC-positive EVs. Here we correlated the cellular response to lipid-based antifibrotic treatment to the relative presence of a candidate protein marker associated with the released EVs. Along with providing insights into polyenylphosphatidylcholines treatments, our findings pave the way for precise and less invasive diagnostic analyses of hepatic fibrogenesis.