Nano-Graphene Oxide-Promoted Epithelial–Mesenchymal Transition of Human Retinal Pigment Epithelial Cells through Regulation of Phospholipase D Signaling

Nano-graphene oxide (Nano-GO) is an extensively studied multifunctional carbon nanomaterial with attractive applications in biomedicine and biotechnology. However, few studies have been conducted to assess the epithelial-to-mesenchymal transition (EMT) in the retinal pigment epithelium (RPE). We aimed to determine whether Nano-GO induces EMT by regulating phospholipase D (PLD) signaling in human RPE (ARPE-19) cells. The physicochemical characterization of Nano-GO was performed using a Zetasizer, X-ray diffraction, Fourier-transform infrared spectroscopy, and transmission electron microscopy. RPE cell viability assays were performed, and the migratory effects of RPE cells were evaluated. RPE cell collagen gel contraction was also determined. Intracellular reactive oxygen species (ROS) levels were determined by fluorescence microscopy and flow cytometry. Immunofluorescence staining and western blot analysis were used to detect EMT-related protein expression. Phospholipase D (PLD) enzymatic activities were also measured. Nano-GO significantly enhanced the scratch-healing ability of RPE cells, indicating that the RPE cell migration ability was increased. Following Nano-GO treatment, the RPE cell penetration of the chamber was significantly promoted, suggesting that the migratory ability was strengthened. We also observed collagen gel contraction and the generation of intracellular ROS in RPE cells. The results showed that Nano-GO induced collagen gel contraction and intracellular ROS production in RPE cells. Moreover, immunofluorescence staining and western blot analysis revealed that Nano-GO significantly regulated key molecules of EMT, including epithelial-cadherin, neural-cadherin, α-smooth muscle actin, vimentin, and matrix metalloproteinases (MMP-2 and MMP-9). Interestingly, Nano-GO-induced RPE cell migration and intracellular ROS production were abrogated in PLD-knockdown RPE cells, indicating that PLD activation played a crucial role in the Nano-GO-induced RPE EMT process. We demonstrate for the first time that Nano-GO promotes RPE cell migration through PLD-mediated ROS production. We provide preliminary evidence to support the hypothesis that Nano-GO has adverse health effects related to RPE damage.

LINC00084/miR-204/ZEB1 Axis Mediates Myofibroblastic Differentiation Activity in Fibrotic Buccal Mucosa Fibroblasts: Therapeutic Target for Oral Submucous Fibrosis

Oral submucosal fibrosis (OSF) is a precancerous condition in the oral cavity and areca nut consumption has been regarded as one of the etiologic factors implicated in the development of OSF via persistent activation of buccal mucosal fibroblasts (BMFs). It has been previously reported that an epithelial to mesenchymal transition (EMT) factor, ZEB1, mediated the areca nut-associated myofibroblast transdifferentiation. In the current study, we aimed to elucidate how areca nut affected non-coding RNAs and the subsequent myofibroblast activation via ZEB1. We found that long non-coding RNA LINC00084 was elicited in the BMFs treated with arecoline, a major alkaloid of areca nut, and silencing LINC00084 prevented the arecoline-induced activities (such as collagen gel contraction, migration, and wound healing capacities). The upregulation of LINC00084 was also observed in the OSF tissues and fibrotic BMFs (fBMFs), and positively correlated with several fibrosis factors. Moreover, we showed knockdown of LINC00084 markedly suppressed the myofibroblast features in fBMFs, including myofibroblast phenotypes and marker expression. The results from the luciferase reporter assay confirmed that LINC00084 acted as a sponge of miR-204 and miR-204 inhibited ZEB1 by directly interacting with it. Altogether, these findings suggested that the constant irritation of arecoline may result in upregulation of LINC00084 in BMFs, which increased the ZEB1 expression by sequestering miR-204 to induce myofibroblast transdifferentiation.

Tropomyosin 2.1 collaborates with fibronectin to promote TGF-β1-induced contraction of human lung fibroblasts

Many lung diseases are characterized by fibrosis, leading to impaired tissue patency and reduced lung function. Development of fibrotic tissue depends on two-way interaction between the cells and the extra-cellular matrix (ECM). Concentration-dependent increased stiffening of the ECM is sensed by the cells, which in turn increases intracellular contraction and pulling on the matrix causing matrix reorganization and further stiffening. It is generally accepted that the inflammatory cytokine growth factor β1 (TGF-β1) is a major driver of lung fibrosis through the stimulation of ECM production. However, TGF-β1 also regulates the expression of members of the tropomyosin (Tm) family of actin associating proteins that mediate ECM reorganization through intracellular-generated forces. Thus, TGF-β1 may mediate the bi-directional signaling between cells and the ECM that promotes tissue fibrosis. Using combinations of cytokine stimulation, mRNA, protein profiling and cellular contractility assays with human lung fibroblasts, we show that concomitant induction of key Tm isoforms and ECM by TGF-β1, significantly accelerates fibrotic phenotypes. Knocking down Tpm2.1 reduces fibroblast-mediated collagen gel contraction. Collectively, the data suggest combined ECM secretion and actin cytoskeleton contractility primes the tissue for enhanced fibrosis. Our study suggests that Tms are at the nexus of inflammation and tissue stiffening. Small molecules targeting specific Tm isoforms have recently been designed; thus targeting Tpm2.1 may represent a novel therapeutic target in lung fibrosis.

Abstract 348: Phorbol 12-myristate 13-acetate Induces Dedifferentiation of Cardiac Myofibroblasts to Fibroblasts and Other Unidentified Type of Cells

Cardiac fibrosis plays an essential role in cardiac pathogenic processes that occur as a result of myocardial infarction or hypertrophic cardiomyopathy. The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in the activation and progression of cardiac fibrosis. TGFβ is one of the essential molecules that promote transition of fibroblasts to myofibroblasts. Reversal of formed myofibroblasts to fibroblasts remains incompletely understood. Phorbol 12-Myristate 13-Acetate (PMA) regulates metabolism and functions of multiple cells via PKC activation mostly. To study effects of PMA on differentiation of de novo formed cardiac myofibroblasts, human cardiac fibroblasts were utilized. Human cardiac fibroblasts (HCF) cultured in fibroblast medium (FM)-2 were converted into myofibroblasts in the presence of 2 ng/mL of TGF-β1 for 48 hours. Expression of α-SMA, the biomarker of myofibroblasts, and FSP1, the biomarker of fibroblasts, was detected using Western blot and immunofluorescence. Collagen gel contraction induced by fibroblasts was determined as well. TGF-β1 increased the expression of α-SMA and reduced the expression of FSP1. Distinct cellular morphology changes in the shape and size of HCF were observed after incubation with TGF-β1 for 48 hours. To investigate effect of PMA on dedifferentiation of formed myofibroblasts, these TGF-β1-pretreated cells were divided into four groups for additional 48 hours incubation: PMA groups (10, 50, and 100 ng/mL) or DMSO (vehicle control). Both 50 and 100 ng/mL of PMA reduced the expression of α-SMA but only 100 ng/mL of PMA increased the expression of FSP1. The shape and size of cells changed after treatment with PMA. PMA also reduced TGF-β1-induced collagen gel contraction (P<0.05, compared to DMSO group). These data indicated that PMA can reverse the differentiation of de novo formed human cardiac myofibroblasts induced by TGF-β1 to fibroblasts and other unidentified type of cells. Although the mechanism of dedifferentiation remains to be identified, the novel finding of this study shed light on future development of agents to treat fibrotic diseases.

SAT0292 INTEGRATIVE TRANSCRIPTOMIC AND FUNCTIONAL ANALYSIS REVEALS A ROLE OF DIMETHYL-Α-KETOGLUTARATE IN TGFΒ-DRIVEN CYTOSKELETON REGULATION AND MYOFIBROBLAST DIFFERENTIATION

Background:Myofibroblasts are the orchestrators of aberrant extracellular matrix (ECM) remodelling in fibrosis. Actin cytoskeleton is a central hub that integrates mechanical signals to promote myofibroblast differentiation and ECM remodelling. Targeting these pathways could represent a novel antifibrotic strategy. We have recently shown that metabolic intermediate dimethyl-α-ketoglutarate (dm-αKG) blocks TGFβ-driven myofibroblast differentiation in dermal fibroblasts (DF).Objectives:To investigate the mechanisms by which dm-αKG regulates TGFβ-driven myofibroblast differentiation and inflammatory responses in DF.Methods:DF from healthy controls and patients with systemic sclerosis (SSc) were treated with TGFβ (10 ng/ml) and/or dm-αKG (6 mM) for 24h, 48h and 72h. RNA sequencing (Ilumina 2000, n=3 per experimental group) was followed by the analysis of differentially expressed genes (DeSEQ2, log2 fold ≥ |0.5|, padj< 0.01), pathway enrichment analysis (GO terms) and supervised PCA analysis (ClustVis). Protein amounts (fibronectin, αSMA, IL-6), cell contraction and apoptosis were measured with Western blot (n=6), ELISA (n=4), collagen gel contraction assay (n=4) and real time Annexin V assay (n=6). Significance (p<0.05) was determined by one-sample t-test or ANOVA with Tukey’s correction for multiple comparisons.Results:TGFβ (24h) altered the expression of 4076 genes in DF as determined by RNA-seq, among which 1864 genes were upregulated. The upregulated genes were enriched in GO biological processes/molecular functions/cellular compartments related to ECM organization (p=1e-07), Wnt signalling (p=5e-06), actin binding (p=3e-07), focal adhesion (p=1e-10), stress fibers (p=3e-07) and actin cytoskeleton (p= 3e-06). Dm-αKG altered the expression of 589 genes in TGFβ-treated DF compared to TGFβ only. The most downregulated pathways in DF treated with dm-αKG + TGFβ compared to TGFβ only included actin binding (p=5e-05), muscle contraction (p=0.001), ECM organization (p=0.008), focal adhesion (p=0.01), Z disk (p=0.01) and stress fibers (p=0.03). Specifically, dm-aKG significantly (p<0.01, log2>-0.5) decreased the expression of many TGFβ-induced genes involved in actin organization and focal adhesion (NEXN, FRMD5, ANTXR1, ACTC1, LIMCH1, SORBS2, TGM2, CSRP2, CAP2, LMO7, FZD2), muscle contraction (SNTB1, LMOD1, ANKRD1, SULF1, JPH2, CAVIN4, OXTR, DYSF, FBXO32) and ECM organization (COL10A1, COL11A1, HAPLN1, MMP14, MMP3, SPINT2, GREM1, MATN3, ADAMTS4). The PCA analysis revealed that the experimental treatment (PC1, Fig 1A) accounted for 61% variability in the expression of these genes, while 19% was attributed to interdonor variability (PC2). Dm-αKG diminished TGFβ-induced production of αSMA protein (72h, p=0.02, mean O.D. ± SD in TGFβ + dm-αKG vs. TGFβ: 0.34 ± 0.38 vs. 3.1 ± 2.3) and repressed TGFβ-driven secretion of fibronectin protein (72h, p=0.047, 0.5 ± 0.1 vs. 1.2 ± 0.6). Dm-αKG reduced the contractile capacity of TGFβ-stimulated DF in collagen gel contraction assay (p=0.003, 0 vs. 67.1 ± 5.4%). Additionally, dm-αKG decreased TGFβ-driven production of IL-6 transcripts (24h, p=0.05, 2.9 ± 0.6 vs 1.9 ± 0.3) and protein (24h, p=0.0005, 5.9 ± 1.2 vs 3 ± 0.7, Fig 1B), but did not increase the apoptosis of DF (24h, 48h, 72h).Fig 1.A Supervised PCA analysis of RNA-seq data. B. IL-6 secretion (ELISA).Conclusion:Dm-αKG counteracted TGFβ-induced myofibroblast differentiation by regulating the cytoskeleton organization and ECM dynamics in DF and blocked the TGFβ-induced IL-6 production. This closely links metabolism to inflammatory and pro-fibrotic responses in DF. Therefore, regulating intracellular αKG might offer a novel strategy in combating the inflammatory and fibrotic stages of skin fibrosis in SSc.Acknowledgments:This work was supported by a research grant from FOREUM Foundation for Research in Rheumatology.Disclosure of Interests:Blaž Burja: None declared, Gabriela Kania: None declared, Matija Tomsic: None declared, Snežna Sodin-Šemrl: None declared, Oliver Distler Grant/research support from: Grants/Research support from Actelion, Bayer, Boehringer Ingelheim, Competitive Drug Development International Ltd. and Mitsubishi Tanabe; he also holds the issued Patent on mir-29 for the treatment of systemic sclerosis (US8247389, EP2331143)., Consultant of: Consultancy fees from Actelion, Acceleron Pharma, AnaMar, Bayer, Baecon Discovery, Blade Therapeutics, Boehringer, CSL Behring, Catenion, ChemomAb, Curzion Pharmaceuticals, Ergonex, Galapagos NV, GSK, Glenmark Pharmaceuticals, Inventiva, Italfarmaco, iQvia, medac, Medscape, Mitsubishi Tanabe Pharma, MSD, Roche, Sanofi and UCB, Speakers bureau: Speaker fees from Actelion, Bayer, Boehringer Ingelheim, Medscape, Pfizer and Roche, Katja Lakota: None declared, Mojca Frank-Bertoncelj: None declared

Inhibition of GPR91 Reduces Inflammatory Mediators Involved in Active Labor in Myometrium

Problem. Some G-protein-coupled receptors (GPCRs) are regulators of inflammation, yet the role of the GPRC, GPR91, is unknown in human myometrium during the processes of human labor and delivery, a major inflammatory event. Method of Study. GPR91 mRNA expression was assessed using RT-qPCR in myometrium obtained from women at term Caesarean section in the absence of labor and during active spontaneous labor and in a mouse model of inflammation-induced preterm labor. Human primary myometrial cells were used to determine the effect of proinflammatory mediators on GPR91 and the effect of GPR91 siRNA on prolabor mediators. Statistical significance was ascribed to a P<0.05. Results. GPR91 mRNA expression was significantly higher in myometrium from women during term spontaneous labor compared to no labor. Likewise, in mice, GPR91 mRNA expression was significantly upregulated in myometrium during inflammation-induced preterm labor compared to preterm no labor. In myometrial cells, IL1B and TNF significantly increased GPR91 mRNA expression. Knockdown of GPR91 by siRNA in myometrial cells significantly suppressed the secretion and/or expression of IL1B- and TNF-induced proinflammatory cytokines (GM-CSF, IL1A, IL1B, and IL6) and chemokines (CXCL8 and CCL2), myometrial contractility (expression of the contraction-associated proteins PTGFR and CX43, secretion of the uterotonic PGF2α, and in situ collagen gel contraction), and the transcription factor NF-κB. Conclusion. Our findings demonstrate that GPR91 is involved in the genesis of proinflammatory and prolabor mediators induced by IL1B or TNF and collectively suggest that GPR91 may contribute to augmentation of the labor processes.

P168 Modelling macrophage-fibroblast interaction in scleroderma to evaluate new treatments

Background Alternatively-activated M2-like macrophages are known to express CD206 and are thought to have an important role in pathological fibrosis in scleroderma through stimulation of fibroblasts. RP peptides are 10-12 mer synthetic peptides which have been developed to engage with human macrophages via CD206, but the selectivity of this receptor for macrophages requires clarification. In this study, we investigate the relative expression levels of CD206 in SSc macrophages and fibroblasts, and explore two approaches in modelling macrophage-fibroblast interactions to evaluate the efficacy of the RP peptides. Methods Macrophages were derived from peripheral blood mononuclear cells by culture of buffy layer cells in RPMI supplemented by M-CSF (4ng/ml) for 7 days. Skin fibroblasts were obtained by explant culture of 4 mm punch biospies from the involved forearm skin of SSc patients and healthy controls, maintained in DMEM with 10-% FCS and studied at passage 3-5. Stimulation with TGFβ (4ng/ml) was used to further enhance the activation state of fibroblasts. Macrophages and skin fibroblast lysates were evaluated using qPCR to determine the level of expression of CD206. Macrophages were co-cultured with fibroblasts within respective monolayers on 50kPa gels (models stiff scleroderma skin) for four days with or without the peptide inhibitors. Collagen mRNA expression from the cellular monolayers were quantified by qPCR and Western blot. In the second approach, macrophages and fibroblasts were co-cultured together with or without the peptide inhibitors in a collagen gel contraction assay. The gel was weighed and imaged after 48 hours of incubation and the collagen gel contraction was then quantified as a measure of fibrotic activity. Results Expression of CD206 was specific to macrophages and not seen in fibroblast cultures (relative expression level in macrophages 26.63 versus 0.004 in unstimulated fibroblasts versus 0.04 in TGFβ-stimulated fibroblasts. Fibroblasts co-cultured with macrophages in monolayers show increased CTGF and collagen mRNA by qPCR. There was a dose response reduction in CTGF and collagen mRNA in assays cultured with the RP peptide inhibitor. CTGF mRNA was positively correlated with CD206 expression (r2=0.81). Co-culture of SSc macrophages led to enhanced contraction of collagen gels (0.1g in macrophages, 0.08g in fibroblasts, 0.062 for fibroblasts co-cultured with macrophages) which was fully reversed by the RP peptide treatment (0.1g). Conclusion SSc macrophages express CD206 which has potential as a biomarker of ongoing fibrotic activity whereas fibroblasts express very minimal levels of CD206 even when stimulated by the pro-fibrotic TGFβ. RP peptides that are specific for CD206 on macrophages led to a suppression of the macrophage signature and inhibition of the pro-fibrotic cross talk in macrophages and fibroblasts in both monolayer and 3D collagen gels. Both approaches of evaluating macrophage-fibroblast interactions in scleroderma are viable approaches to evaluate new treatments. Disclosures L. Lei None. B. Abdi None. A. Mujkavnovic None. X. Shi Wen None. H. Lopez Shareholder/stock ownership; Scientific director at Riptide Biosciences. R. Stratton None.