Inhibition Of Myofibroblast Differentiation Research Articles

Taraxerone exerts antipulmonary fibrosis effect through Smad signaling pathway and antioxidant stress response in a Sirtuin1-dependent manner.

Idiopathic pulmonary fibrosis treatments are limited, often with severe side effects, highlighting the need for novel options. Taraxerone has diverse biomedical properties, but its mechanism remains unclear. This study investigates taraxerone's impact and the mechanisms involved in bleomycin-induced pulmonary fibrosis in mice. After establishing a pulmonary fibrosis mouse model, taraxerone was intraperitoneally injected continuously for 14-28 days. The in vivo antifibrotic and antioxidative stress effects of taraxerone were assessed. In vitro, the influence of taraxerone on transforming growth factor-β1-induced myofibroblast transformation and oxidative stress was investigated. Subsequently, quantitative polymerase chain reaction screened the histone deacetylase and Sirtuin family, and taraxerone's effects on SIRT1 were assessed. After SIRT1 siRNA treatment, changes in myofibroblast transformation and antioxidant capacity in response to taraxerone were observed. Acetylation and phosphorylation levels of Smad3 were evaluated. We also examined the binding levels of SIRT1 with Pho-Smad3 and Smad3, as well as the nuclear localization of Smad2/3. EX527 confirmed SIRT1's in vivo action in response to taraxerone. In vitro experiments suggested that taraxerone inhibited myofibroblast differentiation by activating SIRT1 and reducing oxidative stress. We also observed a new interaction between SIRT1 and the Smad complex. Taraxerone activates SIRT1, enabling it to bind directly to Smad3. This leads to reduced Smad complex phosphorylation and limited nuclear translocation. As a result, the transcription of fibrotic factors is reduced. In vivo validation confirms taraxerone's SIRT1-mediated antifibrotic effectiveness. This suggests that targeting SIRT1-mediated inhibition of myofibroblast differentiation could be a key strategy in taraxerone-based therapy for pulmonary fibrosis.

Supramolecular Nanofibers Ameliorate Bleomycin-Induced Pulmonary Fibrosis by Restoring Autophagy.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease, with limited therapeutic options available. Impaired autophagy resulting from aberrant TRB3/p62 protein-protein interactions (PPIs) contributes to the progression of IPF. Restoration of autophagy by modulating the TRB3/p62 PPIs has rarely been reported for the treatment of IPF. Herein, peptide nanofibers are developed that specifically bind to TRB3 protein and explored their potential as a therapeutic approach for IPF. By conjugating with the self-assembling fragment (Ac-GFFY), a TRB3-binding peptide motif A2 allows for the formation of nanofibers with a stable α-helix secondary structure. The resulting peptide (Ac-GFFY-A2) nanofibers exhibit specific high-affinity binding to TRB3 protein in saline buffer and better capacity of cellular uptake to A2 peptide. Furthermore, the TRB3-targeting peptide nanofibers efficiently interfere with the aberrant TRB3/p62 PPIs in activated fibroblasts and fibrotic lung tissue of mice, thereby restoring autophagy dysfunction. The TRB3-targeting peptide nanofibers inhibit myofibroblast differentiation, collagen production, and fibroblast migration in vitro is demonstrated, as well as bleomycin-induced pulmonary fibrosis in vivo. This study provides a supramolecular method to modulate PPIs and highlights a promising strategy for treating IPF diseases by restoring autophagy.

A quantitative review of nanotechnology-based therapeutics for kidney diseases.

Kidney-specific nanocarriers offer a targeted approach to enhance therapeutic efficacy and reduce off-target effects in renal treatments. The nanocarriers can achieve organ or cell specificity via passive targeting and active targeting mechanisms. Passive targeting capitalizes on the unique physiological traits of the kidney, with factors like particle size, charge, shape, and material properties enhancing organ specificity. Active targeting, on the other hand, achieves renal specificity through ligand-receptor interactions, modifying nanocarriers with molecules, peptides, or antibodies for receptor-mediated delivery. Nanotechnology-enabled therapy targets diseased kidney tissue by modulating podocytes and immune cells to reduce inflammation and enhance tissue repair, or by inhibiting myofibroblast differentiation to mitigate renal fibrosis. This review summarizes the current reports of the drug delivery systems that have been tested in vivo, identifies the nanocarriers that may preferentially accumulate in the kidney, and quantitatively compares the efficacy of various cargo-carrier combinations to outline optimal strategies and future research directions. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Dihydroartemisinin (DHA) inhibits myofibroblast differentiation through inducing ferroptosis mediated by ferritinophagy

Idiopathic pulmonary fibrosis (IPF) is caused by persistent micro-injuries and aberrant repair processes. Myofibroblast differentiation in lung is a key event for abnormal repair. Dihydroartemisinin(DHA), a well-known anti-malarial drug, have been shown to alleviate pulmonary fibrosis, but its mechanism is not clear. Ferroptosis is involved in the pathgenesis of many diseases, including IPF. Ferritinophagy is a form of cellular autophagy which regulates intracellular iron homeostasis. The function of DHA on myofibroblasts differentiation of pulmonary and whether related with ferroptosis and ferritinophagy are unknown now. Using human fetal lung fibroblast 1(HFL1) cell line and the qRT-PCR, immunofluorescent and Western blotting techniques, we found that after TGF-β1 treatment, the levels of ɑ-SMA expression and ROS increased; the mRNA and protein levels of FTH1 and NCOA4, the content of Fe2+ and 4-HNE increased significantly at 6h, then gradually reduced with time. After DHA treatment, FHL1 cells appeared ferroptosis; the levels of α-SMA mRNA and protein reduced and the levels of ROS and 4-HNE increased; the Fe2+ levels decreased sharply at 6h, then increased with time, and were higher than normal since 24h; the mRNA and protein levels of FTH1 and NCOA4 decreased, exhibited a downward trend. These results show that Fe2+, ROS and lipid peroxidation are involved in and ferritinophagy is inhibited during fibroblast-to-myofibroblast differentiation; The depletion of Fe2+ at early stage induced by DHA treatment triggers the ferritinophagy in HFL1 cells, leading to degradation of FTH1 and NCOA4 and following increase of Fe2+ levels. DHA may inhibit the fibroblast-to-myofibroblast differentiation through inducing ferroptosis mediated by ferritinophagy.

Repositioning of ezetimibe for the treatment of idiopathic pulmonary fibrosis.

We previously identified ezetimibe, an inhibitor of Niemann-Pick C1-like intracellular cholesterol transporter 1 and European Medicines Agency-approved lipid-lowering agent, as a potent autophagy activator. However, its efficacy against pulmonary fibrosis has not yet been evaluated. This study aimed to determine whether ezetimibe has therapeutic potential against idiopathic pulmonary fibrosis. Primary lung fibroblasts isolated from both humans and mice were employed for mechanistic in vitro experiments. mRNA sequencing of human lung fibroblasts and gene set enrichment analysis were performed to explore the therapeutic mechanism of ezetimibe. A bleomycin-induced pulmonary fibrosis mouse model was used to examine in vivo efficacy of the drug. Tandem fluorescent-tagged microtubule-associated protein 1 light chain 3 transgenic mice were used to measure autophagic flux. Finally, the medical records of patients with idiopathic pulmonary fibrosis from three different hospitals were reviewed retrospectively, and analyses on survival and lung function were conducted to determine the benefits of ezetimibe. Ezetimibe inhibited myofibroblast differentiation by restoring the mechanistic target of rapamycin complex 1-autophagy axis with fine control of intracellular cholesterol distribution. Serum response factor, a potential autophagic substrate, was identified as a primary downstream effector in this process. Similarly, ezetimibe ameliorated bleomycin-induced pulmonary fibrosis in mice by inhibiting mechanistic target of rapamycin complex 1 activity and increasing autophagic flux, as observed in mouse lung samples. Patients with idiopathic pulmonary fibrosis who regularly used ezetimibe showed decreased rates of all-cause mortality and lung function decline. Our study presents ezetimibe as a potential novel therapeutic for idiopathic pulmonary fibrosis.

Aloe vera peel-derived nanovesicles display anti-inflammatory properties and prevent myofibroblast differentiation.

Aloe vera (AV) is a medicinal plant, most known for its beneficial effects on a variety of skin conditions. Its known active compounds include carbohydrates and flavonoids such as quercetin and kaempferol, among others. In the past decade, plant nanovesicles (NVs) have gained considerable interest as interkingdom communicators, presenting an opportunity for clinical standardization of natural products. In this study, we aimed to assess the potential of AVpNVs for the treatment of burn wounds. AVpNVs were isolated and characterized regarding vesicle yield (nanoparticle tracking analysis) and structure (transmission electron microscopy and atomic force microscopy), as well as their protein content with proteomics. We assessed key characteristics for treating burn wounds in vitro, such as the anti-inflammatory potential in LPS-stimulated macrophages and keratinocytes, and the effect of AVpNVs on myofibroblast differentiation and contraction. AVpNVs presented a homogenous NV population, vesicular shape, and NV-associated protein markers. AVpNVs significantly decreased the secretion of pro-inflammatory cytokines TNFα, IL-1β, and IL-6. Furthermore, AVpNVs inhibited myofibroblast differentiation and significantly decreased their contractile potential in collagen matrices. Observed effects were linked to proteins identified in the isolates through proteomics analysis. AVpNVs displayed characteristics as an inflammatory modulator, while simultaneously diminishing myofibroblast differentiation and contraction. Novel strategies for burn wound treatment seek to decrease scarring on a cellular and molecular level in the early stages of wound healing, which makes AVpNVs a promising candidate for future plant-vesicle-based treatments.

Dexmedetomidine alleviates pulmonary fibrosis through the ADORA2B-Mediated MAPK signaling pathway

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronically progressive fibrotic pulmonary disease characterized by an uncertain etiology, a poor prognosis, and a paucity of efficacious treatment options. Dexmedetomidine (Dex), an anesthetic-sparing alpha-2 adrenoceptor (α2AR) agonist, plays a crucial role in organ injury and fibrosis. However, the underlying mechanisms of IPF remain unknown.MethodsIn our study, the role of Dex in murine pulmonary fibrosis models was determined by Dex injection intraperitoneally in vivo. Fibroblast activation and myofibroblast differentiation were assessed after Dex treatment in vitro. The activation of MAPK pathway and the expression of Adenosine A2B receptor (ADORA2B) were examined in lung myofibroblasts. Moreover, the role of ADORA2B in Dex suppressing myofibroblast differentiation and pulmonary fibrosis was determined using the ADORA2B agonist BAY60-6583.ResultsThe results revealed that Dex could inhibit Bleo-induced pulmonary fibrosis in mice. In vitro studies revealed that Dex suppressed TGF-β-mediated MAPK pathway activation and myofibroblast differentiation. Furthermore, Dex inhibits myofibroblast differentiation and pulmonary fibrosis via downregulating ADORA2B expression.ConclusionsOur findings suggest Dex as a potential therapeutic agent for pulmonary fibrosis. Dex may alleviate lung fibrosis and myofibroblast differentiation through the ADORA2B-mediated MAPK signaling pathway.

Abstract P2133: Preservation & Vascularization Of Cardiac Extracellular Matrix After Acute Myocardial Infarction

Following myocardial infarction (MI), the upregulated matrix metalloproteinases (MMPs) especially MMP-2/9 lead to the degradation of cardiac extracellular matrix (ECM), leading to impaired cardiac function. Therefore, preservation of cardiac ECM by decreasing MMP-2/9 acitivity will increase cardiac function. Meanwhile, vascularization is critical for cardiac ECM as otherwise its structure and composition change over time. In this work, a non-toxic peptide CTTHWGFTLC (CTT) that specifically inhibits MMP-2/9, and a pro-angiogenic basic fibroblast growth factor (bFGF) were used to preserve and vascularize the cardiac ECM. A fast gelling, degradable, and reactive oxygen species (ROS)-scavenging hydrogel was developed to deliver CTT and bFGF to the infarcted hearts. We evaluated the efficacy of the delivery system in preserving and vascularizing cardiac ECM, and improving cardiac function. The combination of CTT and bFGF synergistically promoted tube formation of HUVECs compared to the treatment with CTT or bFGF (**p<0.01). Interestingly, both CTT and bFGF inhibited myofibroblast differentiation of cardiac fibroblasts. The combination of CTT and bFGF further significantly decreased the differentiation. After 28 days of implantation of the CTT/bFGF/Gel, the wall thickness was significantly increased compared to the CTT/Gel, bFGF/Gel, Gel only and MI. The released CTT and bFGF preserved cardiac ECM, decreased cardiac fibrosis, and stimulated angiogenesis in the infarcted area, leading to a significant increase of cardiac function.

Aligned lovastatin-loaded electrospun nanofibers regulate collagen organization and reduce scar formation.

Excessive scar formation caused by cutaneous injury leads to pruritus, pain, contracture, dyskinesia, and unpleasant appearance. Functional wound dressings are designed to accelerate wound healing and reduce scar formation. In this study, we fabricated aligned or random polycaprolactone/silk fibroin electrospun nanofiber membranes with or without lovastatin loading, and then evaluated their scar-inhibitory effects on wounds under a specific tension direction. The nanofiber membranes exhibited good controlled-release performance, mechanical properties, hydrophilicity, and biocompatibility. Furthermore, nanofibers' perpendicular placement to the tension direction of the wound most effectively reduced scar formation (the scar area decreased by 66.9%) and promotedskin regeneration in vivo. The mechanism was associated with aligned nanofibers regulated collagen organization in the early stage of wound healing. Moreover, lovastatin-loaded nanofibers inhibited myofibroblast differentiation and migration. Both tension direction-perpendicular topographical cues and lovastatin synergistically inhibited mechanical transduction and fibrosis progression, further reducing scar formation. In summary, our study may provide an effective scar prevention strategy in which individualized dressings can be designed according to the local mechanical force direction of patients' wounds, and the addition of lovastatin can further inhibit scar formation. STATEMENT OF SIGNIFICANCE: In vivo, cells and collagen are always arranged parallel to the tension direction. However, the aligned topographic cues themselves promote myofibroblast differentiation and exacerbate scar formation. Electrospun nanofibers' perpendicular placement to the tension direction of the wound most effectively reduces scar formation and promotes skin regeneration in vivo. The mechanism is associated with tension direction-perpendicular nanofibers reregulate collagen organization in the early stage of wound healing. In addition, tension direction-perpendicular topographical cue and lovastatin could inhibit mechanical transduction and fibrosis progression synergistically, further reducing scar formation. This study proves that combining topographical cues of wound dressing and drugs would be a promising therapy for clinical scar management.

Mitochondrial folate pathway regulates myofibroblast differentiation and silica-induced pulmonary fibrosis

BackgroundSilica-induced pulmonary fibrosis (silicosis) is a diffuse interstitial fibrotic disease characterized by the massive deposition of extracellular matrix in lung tissue. Fibroblast to myofibroblast differentiation is crucial for the disease progression. Inhibiting myofibroblast differentiation may be an effective way for pulmonary fibrosis treatment.MethodsThe experiments were conducted in TGF-β treated human lung fibroblasts to induce myofibroblast differentiation in vitro and silica treated mice to induce pulmonary fibrosis in vivo.ResultsBy quantitative mass spectrometry, we revealed that proteins involved in mitochondrial folate metabolism were specifically upregulated during myofibroblast differentiation following TGF-β stimulation. The expression level of proteins in mitochondrial folate pathway, MTHFD2 and SLC25A32, negatively regulated myofibroblast differentiation. Moreover, plasma folate concentration was significantly reduced in patients and mice with silicosis. Folate supplementation elevated the expression of MTHFD2 and SLC25A32, alleviated oxidative stress and effectively suppressed myofibroblast differentiation and silica-induced pulmonary fibrosis in mice.ConclusionOur study suggests that mitochondrial folate pathway regulates myofibroblast differentiation and could serve as a potential target for ameliorating silica-induced pulmonary fibrosis.

Evaluation of prevention and treatment effects of fibroblast growth factor-21 in BLM-induced pulmonary fibrosis.

Pulmonary fibrosis is a progressive and fatal fibrotic lung disease and associated with a high mortality rate. In the study, the prevention and treatment effects of fibroblast growth factor-21 (FGF-21) in bleomycin (BLM)-induced pulmonary fibrosis were investigated in vivo and vitro. In the prevention of pulmonary fibrosis studies, the results showed that interdict of FGF-21 could reduce the related gene and protein expression levels of pulmonary fibrosis. In addition, FGF-21 significantly reduced both the aggregation of inflammatory cells and deposition of collagen in the lung by histopathology. In therapy of pulmonary fibrosis studies, the results indicated that treatment with FGF-21 resulted in an amelioration of the pulmonary fibrosis in mice with reductions of the pathological score, collagen deposition and transforming growth factor (TGF)-β and α-smooth muscle actin (α-SMA) expressions in the lung tissues at fibrotic stage, and late administration was also able to reduce the degree of pulmonary fibrosis and even better than these in the prevention group. Furthermore, BLM-induced THP-1 macrophage model was verified using FGF-21; the result showed that FGF-21 decreased the related gene expression level of pulmonary fibrosis. FGF-21 may have preventive and therapeutic effects on BLM-induced pulmonary fibrosis via inhibiting myofibroblast differentiation and inflammatory. Thus, FGF-21 represents a potential drug for the prevention and treatment of pulmonary fibrosis.

Phosphodiesterase type 10A inhibitor attenuates lung fibrosis by targeting myofibroblast activation

Pulmonary fibrosis (PF) is a fatal and irreversible respiratory disease accompanied by excessive fibroblast activation. Previous studies have suggested that cAMP signaling pathway and cGMP-PKG signaling pathway are continuously down-regulated in lung fibrosis, whereas PDE10A has a specifically expression in fibroblasts/myofibroblasts in lung fibrosis. In this study, we demonstrated that overexpression of PDE10A induces myofibroblast differentiation, and papaverine, as a PDE10A inhibitor used for vasodilation, inhibits myofibroblast differentiation in human fibroblasts, Meanwhile, papaverine alleviated bleomycin-induced pulmonary fibrosis and amiodarone-induced oxidative stress, papaverine downregulated VASP/β-catenin pathway to reduce the myofibroblast differentiation. Our results first demonstrated that papaverine inhibits TGFβ1-induced myofibroblast differentiation and lung fibrosis by VASP/β-catenin pathway.

MiR-27a-3p alleviates lung transplantation-induced bronchiolitis obliterans syndrome (BOS) via suppressing Smad-mediated myofibroblast differentiation and TLR4-induced dendritic cells maturation

BackgroundBronchiolitis obliterans syndrome (BOS), induced by a chronic rejection, remains a significant obstacle for end-stage lung diseases after lung transplantation. We have previously determined that the small non-coding mRNA (miRNA) miR-27a-3p maintained the immature state of dendritic cells (DCs) via the interleukin 10 (IL-10)-dependent regulatory pathway. Such status helped in preventing rejection and alleviating BOS. The present study explored mechanisms how miR-27a-3p may suppress the fibrosis as well as the maturation of DCs, ultimately attenuating BOS in vitro and in vivo. Methods/ResultsIn our tracheal transplantation mouse model, the expression of Smad2, Smad4, and αSMA were significantly decreased in the miR-27a-3p-transfected DCs (p < 0.0001, p = 0.0006, and p = 0.0002 respectively). Moreover, the expression of fibrosis markers (α-SMA, collagen I, and Fn) were potently inhibited in the miR-27a-3p-transfected NIH-3 T3 cells (p < 0.0001, p = 0.00148, and p < 0.0001 respectively). At the same time, reversed results were observed in the inhibitor group (p = 0.0002, p < 0.0001, and p < 0.0001 respectively), indicating that miR-27a-3p could directly inhibit myofibroblast differentiation. Furthermore, in the tracheal transplanted mice, the population of Treg cells was significantly decreased (p < 0.0001). In contrast, Th17 cells were down-regulated in the miR-27a-3p-transfected DCs group (p < 0.0001), accompanied by the decreased IL-17 levels (p = 0.0007) and the induction of TGF-β1 and IL-10 (p < 0.0001 and p = 0.0016 respectively). Further mechanistic studies indicated that miR-27a-3p altered the maturation of DCs by targeting TLR4 and IRAK (p < 0.0001 and p = 0.0002 respectively). ConclusionsOur study suggests that miR-27a-3p selectively blocked the TGF-β1/Smad pathways to suppress the myofibroblast differentiation and targeted the TRL4/IRAK4 pathway to restrain DCs maturation, thus attenuating BOS. Our findings suggest that miR-27a-3p is a potential active molecule on BOS management after lung transplantation.

The Novel Small Molecule BTB Inhibits Pro-Fibrotic Fibroblast Behavior though Inhibition of RhoA Activity.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, interstitial lung disease with a poor prognosis. Although specific anti-fibrotic medications are now available, the median survival time following diagnosis remains very low, and new therapies are urgently needed. To uncover novel therapeutic targets, we examined how biochemical properties of the fibrotic lung are different from the healthy lung. Previous work identified lactate as a metabolite that is upregulated in IPF lung tissue. Importantly, inhibition of the enzyme responsible for lactate production prevents fibrosis in vivo. Further studies revealed that fibrotic lesions of the lung experience a significant decline in tissue pH, likely due to the overproduction of lactate. It is not entirely clear how cells in the lung respond to changes in extracellular pH, but a family of proton sensing G-protein coupled receptors has been shown to be activated by reductions in extracellular pH. This work examines the expression profiles of proton sensing GPCRs in non-fibrotic and IPF-derived primary human lung fibroblasts. We identify TDAG8 as a proton sensing GPCR that is upregulated in IPF fibroblasts and that knockdown of TDAG8 dampens myofibroblast differentiation. To our surprise, BTB, a proposed positive allosteric modulator of TDAG8, inhibits myofibroblast differentiation. Our data suggest that BTB does not require TDAG8 to inhibit myofibroblast differentiation, but rather inhibits myofibroblast differentiation through suppression of RhoA mediated signaling. Our work highlights the therapeutic potential of BTB as an anti-fibrotic treatment and expands upon the importance of RhoA-mediated signaling pathways in the context of myofibroblast differentiation. Furthermore, this works also suggests that TDAG8 inhibition may have therapeutic relevance in the treatment of IPF.

Deletion of the extracellular matrix glycoprotein ADAMTSL3 increases pro-fibrotic signalling and exacerbates heart failure in mice

Abstract Background/Introduction Cardiac fibrosis is a central pathophysiological process accompanying most cardiac diseases including heart failure, and is a predictor of poor outcomes. The fibrotic process is mediated by activated cardiac fibroblasts (CFBs), so-called myofibroblasts, which produce excessive amounts of type I collagen and other ECM molecules. The ADAMTSL family of glycoproteins is upregulated in the cardiac extracellular matrix (ECM) during heart failure, but their function in the heart is unknown. Some ADAMTSL proteins are suggested to regulate the TGFβ, a major driver of myofibroblast activation and cardiac fibrosis. Purpose The purpose of this study was to examine the role of ADAMTSL3 in TGFβ-regulation and heart failure. Methods ADAMTSL3 expression was analysed in failing hearts of patients and mice. To study the functional role of ADAMTSL3 in the failing heart, an Adamtsl3 knock-out (L3-KO) mouse was generated, and WT and L3-KO littermates were subjected to experimental heart failure by aortic banding (AB), or sham surgery, for a total of six weeks. The mice were followed with echocardiography and MRI, and the left ventricles (LVs) were harvested one and six weeks after AB or sham surgery, with molecular analyses performed. To elucidate molecular functions of ADAMTSL3 in vitro, we overexpressed ADAMTSL3 in CFBs producing an extensive ECM. Results We determined that ADAMTSL3 was produced by CFBs and upregulated in failing hearts of patients and mice. The L3-KO mice had a normal cardiac phenotype at baseline, but upon increased LV afterload developed a dilated cardiomyopathy phenotype with increased LV dilation and reduced systolic function from one week post-AB. Furthermore, high mortality was observed in the L3-KO mice post-AB, with 60% vs. 96% survival of WTs over six weeks. At one week post-AB RNA sequencing of LVs revealed 233 differentially expressed genes in L3-KO vs. WT, with Col1a1 and Postn among the most upregulated, suggesting increased fibrosis and TGFβ signalling. Increased canonical TGFβ signalling was confirmed by increased SMAD2/3 phosphorylation and increased Lox expression in L3-KOs six weeks post-AB. In vitro, ADAMTSL3 overexpression in cultured CFBs resulted in reduced collagen synthesis and reduced expression of COL1A1, LOX, CTGF and POSTN, indicating anti-fibrotic properties. Furthermore, ADAMTSL3 inhibited the expression of ACTA2 and SPP1, reduced α-SMA protein by 25%, and reduced proliferation and CFB contraction, suggesting attenuated TGFβ signalling and inhibited myofibroblast differentiation. Conclusions ADAMTSL3 was upregulated in human and mouse heart failure, and served an anti-fibrotic and cardio-protective role in failing mouse hearts. Mechanistically, ADAMTSL3 was produced by CFBs and inhibited myofibroblast differentiation and collagen synthesis through TGFβ in cultured CFBs. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Research Council of Norway

Dexamethasone and olmesartan as potential antiremodelling agents of valvular interstitial cell into myofibroblast

Rheumatic heart disease is a late complication of valvular inflammation caused by rheumatic fever. Studies have shown that the differentiation of valvular interstitial cells (VIC) into fibroblasts plays an important role in valvular remodeling and fibrosis. Various strategies to minimize valvular fibrosis has increased recently. This study aims to analyze the effect of dexamethasone, olmesartan, and its combination in inhibiting TGF-β1-induced VIC differentiation into myofibroblast. In vitro laboratory experimental-posttest only control group design was conducted. Isolated VIC of Oryctolagus cuniculus was pretreated using 2,5 ng/mL of TGF-β1 and divided into groups of dexamethasone (0.1 uM/L), olmesartan (100 nmol/L), and its combination. Inhibition of myofibroblast differentiation was quantified by the expression of α-SMA levels detected by immunofluorescence. Dexamethasone, olmesartan, and its combination administration were significantly reduced TGF-β1-induced VIC differentiation into myofibroblast expressed by α-SMA levels (dexamethasone 6823 ± 1735.3, olmesartan 6683.7 ± 2795.05). Combination of dexamethasone and olmesartan exhibit the most potent inhibition compared to control (5051.87 ± 1612.210 vs 22286.73 ± 2780.2; p &lt; 0.000). In conclusion, dexamethasone, olmesartan, and the combination can significantly reduce the differentiation of VIC into myofibroblasts. The greatest potential is the combined effect of dexamethasone and olmesartan, while dexamethasone and olmesartan have the same potential.

Olmesartan inhibits transdifferentiation of rabbit’s valvular interstitial cells into myofibroblast based on α – smooth muscle actin expression

Recent studies revealed that differentiation of valvular interstitial cell (VIC) into myofibroblasts played an important role in valvular remodeling and fibrosis. Study was performed on in vitro cultured of VIC from New Zealand rabbit (Oryctolagus cuniculus). Isolated VIC was pretreated using 5 ng/mL of TGF-β1 and divided into groups of olmesartan (100 nanomol/L) as short 30 minutes exposure duration and as continuous 72 hours exposure duration. Inhibition of myofibroblast differentiation was quantified by the expression of α-SMA levels detected by immunofluorescence staining. Olmesartan administration either, as short 30 minutes exposure duration or as continuous 72 hours exposure duration, were significantly reduced TGF-β1-induced VIC differentiation into myofibroblast expressed by α-SMA levels compared to control (short 30 minutes exposure duration: 9.18 ±2.25, as continuous 72 hours exposure duration: 10.13 ±0.69, and control 22.29 ±2.78; p &lt; 0.001), but without significant difference between the two treatment groups (p 0.403). Olmesartan both on short and continuous exposure can inhibit the differentiation of valve interstitial cells into myofibroblasts based on the expression of αSMA, but without a significant difference in the inhibitory effect.

Cell-Type-Specific Effects of the Ovarian Cancer G-Protein Coupled Receptor (OGR1) on Inflammation and Fibrosis; Potential Implications for Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by irreversible lung scarring. The pathophysiology is not fully understood, but the working hypothesis postulates that a combination of epithelial injury and myofibroblast differentiation drives progressive pulmonary fibrosis. We previously demonstrated that a reduction in extracellular pH activates latent TGF-β1, and that TGF-β1 then drives its own activation, creating a feed-forward mechanism that propagates myofibroblast differentiation. Given the important roles of extracellular pH in the progression of pulmonary fibrosis, we sought to identify whether pH mediates other cellular phenotypes independent of TGF-β1. Proton-sensing G-protein coupled receptors are activated by acidic environments, but their role in fibrosis has not been studied. Here, we report that the Ovarian Cancer G-Protein Coupled Receptor1 (OGR1 or GPR68) has dual roles in both promoting and mitigating pulmonary fibrosis. We demonstrate that OGR1 protein expression is significantly reduced in lung tissue from patients with IPF and that TGF-β1 decreases OGR1 expression. In fibroblasts, OGR1 inhibits myofibroblast differentiation and does not contribute to inflammation. However, in epithelial cells, OGR1 promotes epithelial to mesenchymal transition (EMT) and inflammation. We then demonstrate that sub-cellular localization and alternative signaling pathways may be responsible for the differential effect of OGR1 in each cell type. Our results suggest that strategies to selectively target OGR1 expression may represent a novel therapeutic strategy for pulmonary fibrosis.

Ogerin mediated inhibition of TGF-β(1) induced myofibroblast differentiation is potentiated by acidic pH.

Transforming growth factor beta (TGF-β) induced myofibroblast differentiation is central to the pathological scarring observed in Idiopathic Pulmonary Fibrosis (IPF) and other fibrotic diseases. Our lab has recently identified expression of GPR68 (Ovarian Cancer Gene Receptor 1, OGR1), a pH sensing G-protein coupled receptor, as a negative regulator of TGF-β induced profibrotic effects in primary human lung fibroblasts (PHLFs). We therefore hypothesized that small molecule activators of GPR68 would inhibit myofibroblast differentiation. Ogerin is a positive allosteric modulator (PAM) of GPR68, inducing a leftward shift of the dose response curve to proton induced signaling. Using PHLFs derived from patients with both non-fibrotic and IPF diagnoses, we show that Ogerin inhibits, and partially reverses TGF-β induced myofibroblast differentiation in a dose dependent manner. This occurs at the transcriptional level without inhibition of canonical TGF-β induced SMAD signaling. Ogerin induces PKA dependent CREB phosphorylation, a marker of Gαs pathway activation. The ability of Ogerin to inhibit both basal and TGF-β induced collagen gene transcription, and induction of Gαs signaling is enhanced at an acidic pH (pH 6.8). Similar findings were also found using fibroblasts derived from dermal, intestinal, and orbital tissue. The biological role of GPR68 in different tissues, cell types, and disease states is an evolving and emerging field. This work adds to the understanding of Gαs coupled GPCRs in fibrotic lung disease, the ability to harness the pH sensing properties of GPR68, and conserved mechanisms of fibrosis across different organ systems.

Tofacitinib May Inhibit Myofibroblast Differentiation from Rheumatoid-Fibroblast-like Synoviocytes Induced by TGF-β and IL-6

During rheumatoid arthritis (RA), the pathogenic role of resident cells within the synovial membrane is suggested, especially for a population frequently referred to as fibroblast-like synoviocytes (FLSs). In this study, we assess the markers of myofibroblast differentiation of RA-FLSs by ex vivo observations and in vitro evaluations following the stimulation with both TGF-β and IL-6. Furthermore, we investigated the possible inhibiting role of tofacitinib, a JAK inhibitor, in this context. Myofibroblast differentiation markers were evaluated on RA synovial tissues by immune-fluorescence or immune-histochemistry. RA-FLSs, stimulated with transforming growth factor (TGF-β) and interleukin-6 (IL-6) with/without tofacitinib, were assessed for myofibroblast differentiation markers expression by qRT-PCR and Western blot. The same markers were evaluated following JAK-1 silencing by siRNA assay. The presence of myofibroblast differentiation markers in RA synovial tissue was significantly higher than healthy controls. Ex vivo, α-SMA was increased, whereas E-Cadherin decreased. In vitro, TGF-β and IL-6 stimulation of RA-FLSs promoted a significant increased mRNA expression of collagen I and α-SMA, whereas E-Cadherin mRNA expression was decreased. In the same conditions, the stimulation with tofacitinib significantly reduced the mRNA expression of collagen I and α-SMA, even if the Western blot did not confirm this finding. JAK-1 gene silencing did not fully prevent the effects of stimulation with TGF-β and IL-6 on these features. TGF-β and IL-6 stimulation may play a role in mediating myofibroblast differentiation from RA-FLSs, promoting collagen I and α-SMA while decreasing E-Cadherin. Following the same stimulation, tofacitinib reduced the increases of both collagen I and α-SMA on RA-FLSs, although further studies are needed to fully evaluate this issue and confirm our results.