Profibrotic Phenotype Research Articles

OA07 Clinical and pathogenic significance of S100A4 overexpression in systemic sclerosis

Abstract Background/Aims Preclinical studies suggest that the S100A4, a Damage Associated Molecular Pattern (DAMP) protein upregulated upon stress or injury, may be a target for antifibrotic therapy. We have studied expression and function of S100A4 in systemic sclerosis (SSc) to further explore its relevance as a driver of fibroblast activation. Methods S100A4 protein concentration was measured by ELISA (Cusabio, USA) in serum of SSc (n = 94) and healthy controls (n = 15). Association of serum level with major organ complications and skin score (mRSS) was determined. S100A4 protein expression in supernatant and cell layer of skin fibroblast cultures from diffuse cutaneous SSc (SScF, n = 6) and healthy controls (NF, n = 6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were used to investigate activation of profibrotic molecular and functional phenotype in SSc and control fibroblasts. Results Median [range] S100A4 (ng/ml) was higher in serum of SSc (89.9 [15.0-240.0]) than healthy controls (71.4 [7.9-131.8]; p = 0.027). There was significant association with SSc-ILD (p = 0.025, n = 55), scleroderma renal crisis (p = 0.026, n = 4) and trend of increase in pulmonary hypertension (p = 0.075, n = 7). There was a trend for S100A4 to fall in longer disease duration (r=-0.18, p = 0.09) and a weak (r = 0.28, p = 0.07) correlation with current mRSS but not peak mRSS in dcSSc. Median [range] S100A4 (ng/ml) was higher in culture supernatants of SScF (4.19 [0.52-8.42]) than NF controls (0.28 [0.02-3.29]; p < 0.0001) and SSc cell layer had increased levels by Western blot analysis (mean [sd] RDU NF = 0.50 [0.91]; SScF 8.16 [2.49]; p < 0.0001). S100A4 induced a fibrotic phenotype in NF with concentration dependent increased in Col1, SMA and CTGF expression, and promotion of migration (scratch wound assay) and 3D type I collagen gel contraction that was attenuated by AX-202. AX-202 also reduced the constitutive profibrotic gene and protein expression phenotype of SScF. The effect of S100A4 was significantly attenuated by SB431524, a specific inhibitor of ALK5 (p < 0.0001 ANOVA) using qPCR assays across treatment groups for Col1, SMA and CTGF. Gene expression data (qPCR) confirmed protein expression. Genome-wide RNAseq analysis identified an S100A4 activated signature in NF overlapping the hallmark gene expression signature of SScF. Thus, 464 differentially expressed genes (FDR<0.001 and FC > 1.5) induced in NF by S100A4 were also constitutively overexpressed in SScF, and significantly downregulated by AX-202. Pathway mapping of these S100A4 dependent transcripts in SSc showed the most significant enriched Kegg® pathways (FDR<0.001) were regulation of stem cell pluripotency (4.6-fold) and metabolic pathways (1.9-fold). Conclusion Our findings provide compelling evidence supporting a profibrotic role for S100A4 in fibroblast activation in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. Studies to elucidate the therapeutic potential of targeting S100A4 in SSc are warranted. Disclosure C.P. Denton: None. S. Xu: None. R.H. Maclean: None. K.E. Clark: None. S. Borchert: Corporate appointments; Employee of ARXX Therapeutics. R. Hussain: Corporate appointments; Employee of ARXX Therapeutics. J. Klingelhöfer: Corporate appointments; Employee of ARXX Therapeutics. J. Hallén: Corporate appointments; Employee of ARXX Therapeutics. V.H. Ong: None.

Tubular cell senescence promotes maladaptive kidney repair and chronic kidney disease after cisplatin nephrotoxicity.

Cisplatin is a widely used chemotherapy drug; however, it induces both acute and chronic kidney diseases (CKD) in patients with cancer. The pathogenesis of cisplatin-induced CKD is unclear, and effective renoprotective approaches are not available. Here, we report that repeated low-dose cisplatin (RLDC) treatment of C57BL/6 mice induced chronic cellular senescence in kidney tubules, accompanied with tubular degeneration and profibrotic phenotype transformation that culminated in maladaptive repair and renal fibrosis. Suppression of tubular senescence by senolytic drugs ABT-263 and Fisetin attenuated renal fibrosis and improved tubular repair, as indicated by restoration of tubular regeneration and renal function. In vitro, RLDC also induced senescence in mouse proximal tubular (BUMPT) cells. ABT-263 eliminated senescent BUMPT cells following RLDC treatment, reversed the profibrotic phenotype of the cells, and increased their clonogenic activity. Moreover, ABT-263 alleviated the paracrine effect of RLDC-treated BUMPT cells on fibroblasts for fibrosis. Consistently, knockdown of p16 suppressed post-RLDC senescence and fibrotic changes in BUMPT cells and alleviated their paracrine effects on renal fibroblast proliferation. These results indicate that persistent induction of tubular senescence plays an important role in promoting cisplatin-induced CKD. Targeting senescent tubular cells may be efficient for improvement of kidney repair and for the prevention and treatment of cisplatin-induced CKD.

Early events marking lung fibroblast transition to profibrotic state in idiopathic pulmonary fibrosis

BackgroundIdiopathic Pulmonary Fibrosis (IPF) is an age-associated progressive lung disease with accumulation of scar tissue impairing gas exchange. Previous high-throughput studies elucidated the role of cellular heterogeneity and molecular pathways in advanced disease. However, critical pathogenic pathways occurring in the transition of fibroblasts from normal to profibrotic have been largely overlooked.MethodsWe used single cell transcriptomics (scRNA-seq) from lungs of healthy controls and IPF patients (lower and upper lobes). We identified fibroblast subclusters, genes and pathways associated with early disease. Immunofluorescence assays validated the role of MOXD1 early in fibrosis.ResultsWe identified four distinct fibroblast subgroups, including one marking the normal-to-profibrotic state transition. Our results show for the first time that global downregulation of ribosomal proteins and significant upregulation of the majority of copper-binding proteins, including MOXD1, mark the IPF transition. We find no significant differences in gene expression in IPF upper and lower lobe samples, which were selected to have low and high degree of fibrosis, respectively.ConclusionsEarly events during IPF onset in fibroblasts include dysregulation of ribosomal and copper-binding proteins. Fibroblasts in early stage IPF may have already acquired a profibrotic phenotype while hallmarks of advanced disease, including fibroblast foci and honeycomb formation, are still not evident. The new transitional fibroblasts we discover could prove very important for studying the role of fibroblast plasticity in disease progression and help develop early diagnosis tools and therapeutic interventions targeting earlier disease states.

Ubiquitin Ligases Siah1a/2 Control Alveolar Macrophage Functions to Limit Carcinogen-Induced Lung Adenocarcinoma.

The ubiquitin ligases Siah1a/2 control proinflammatory signaling, differentiation, and profibrotic phenotypes of alveolar macrophages to suppress lung carcinogenesis.

Extracellular Lactic Acidosis of the Tumor Microenvironment Drives Adipocyte-to-Myofibroblast Transition Fueling the Generation of Cancer-Associated Fibroblasts.

Lactic acidosis characterizes the tumor microenvironment (TME) and is involved in the mechanisms leading to cancer progression and dissemination through the reprogramming of tumor and local host cells (e.g., endothelial cells, fibroblasts, and immune cells). Adipose tissue also represents a crucial component of the TME which is receiving increasing attention due to its pro-tumoral activity, however, to date, it is not known whether it could be affected by the acidic TME. Now, emerging evidence from chronic inflammatory and fibrotic diseases underlines that adipocytes may give rise to pathogenic myofibroblast-like cells through the adipocyte-to-myofibroblast transition (AMT). Thus, our study aimed to investigate whether extracellular acidosis could affect the AMT process, sustaining the acquisition by adipocytes of a cancer-associated fibroblast (CAF)-like phenotype with a pro-tumoral activity. To this purpose, human subcutaneous adipose-derived stem cells committed to adipocytes (acADSCs) were cultured under basal (pH 7.4) or lactic acidic (pH 6.7, 10 mM lactate) conditions, and AMT was evaluated with quantitative PCR, immunoblotting, and immunofluorescence analyses. We observed that lactic acidosis significantly impaired the expression of adipocytic markers while inducing myofibroblastic, pro-fibrotic, and pro-inflammatory phenotypes in acADSCs, which are characteristic of AMT reprogramming. Interestingly, the conditioned medium of lactic acidosis-exposed acADSC cultures was able to induce myofibroblastic activation in normal fibroblasts and sustain the proliferation, migration, invasion, and therapy resistance of breast cancer cells in vitro. This study reveals a previously unrecognized relationship between lactic acidosis and the generation of a new CAF-like cell subpopulation from adipocytic precursor cells sustaining tumor malignancy.

Increased Renal Medullary NOX-4 in Female but Not Male Mice during the Early Phase of Type 1 Diabetes: Potential Role of ROS in Upregulation of TGF-β1 and Fibronectin in Collecting Duct Cells

Chronic diabetes mellitus (DM) can lead to kidney damage associated with increased reactive oxygen species (ROS), proteinuria, and tubular damage. Altered protein expression levels of transforming growth factor-beta 1 (TGF-β1), fibronectin, and renal NADPH oxidase (NOX-4) are associated with the profibrotic phenotype in renal tubular cells. NOX-4 is one of the primary sources of ROS in the diabetic kidney and responsible for the induction of profibrotic factors in collecting duct (CD) cells. The renal medulla is predominantly composed of CDs; in DM, these CD cells are exposed to high glucose (HG) load. Currently there is no published literature describing the expression of these markers in the renal medulla in male and female mice during the early phase of DM, or the role of NOX-4-induced ROS. Our aim was to evaluate changes in transcripts and protein abundances of TGF-β1, fibronectin, and NOX-4 along with ROS levels in renal medullary tissues from male and female mice during a short period of streptozotocin (STZ)-induced type 1 DM and the effect of HG in cultured CD cells. CF-1 mice were injected with or without a single dose of STZ (200 mg/kg) and euthanized at day 6. STZ females showed higher expression of fibronectin and TGF-β1 when compared to control mice of either gender. Interestingly, STZ female mice showed a >30-fold increase on mRNA levels and a 3-fold increase in protein levels of kidney medullary NOX-4. Both male and female STZ mice showed increased intrarenal ROS. In primary cultures of inner medullary CD cells exposed to HG over 48 h, the expression of TGF-β1, fibronectin, and NOX-4 were augmented. M-1 CD cells exposed to HG showed increased ROS, fibronectin, and TGF-β1; this effect was prevented by NOX-4 inhibition. Our data suggest that at as early as 6 days of STZ-induced DM, the expression of profibrotic markers TGF-β1 and fibronectin increases in renal medullary CD cells. Antioxidants mechanisms in male and female in renal medullary tissues seems to be differentially regulated by the actions of NOX-4.

B-Klotho deficiency in hepatic stellate cells (HSCs) prompts inflammation, oxidative stress and a pro-fibrotic phenotype

B-Klotho deficiency in hepatic stellate cells (HSCs) prompts inflammation, oxidative stress and a pro-fibrotic phenotype

Collagen-Specific HSP47+ Myofibroblasts and CD163+ Macrophages Identify Profibrotic Phenotypes in Deceased Hearts With SARS-CoV-2 Infections.

Background Cardiac fibrosis complicates SARS-CoV-2 infections and has been linked to arrhythmic complications in survivors. Accordingly, we sought evidence of increased HSP47 (heat shock protein 47), a stress-inducible chaperone protein that regulates biosynthesis and secretion of procollagen in heart tissue, with the goal of elucidating molecular mechanisms underlying cardiac fibrosis in subjects with this viral infection. Methods and Results Using human autopsy tissue, immunofluorescence, and immunohistochemistry, we quantified Hsp47+ cells and collagen α 1(l) in hearts from people with SARS-CoV-2 infections. Because macrophages are also linked to inflammation, we measured CD163+ cells in the same tissues. We observed irregular groups of spindle-shaped HSP47+ and CD163+ cells as well as increased collagen α 1(I) deposition, each proximate to one another in "hot spots" of ≈40% of hearts after SARS-CoV-2 infection (HSP47+ P<0.05 versus nonfibrotics and P<0.001 versus controls). Because HSP47+ cells are consistent with myofibroblasts, subjects with hot spots are termed "profibrotic." The remaining 60% of subjects dying with COVID-19 without hot spots are referred to as "nonfibrotic." No control subject exhibited hot spots. Conclusions Colocalization of myofibroblasts, M2(CD163+) macrophages, and collagen α 1(l) may be the first evidence of a COVID-19-related "profibrotic phenotype" in human hearts in situ. The potential public health and diagnostic implications of these observations require follow-up to further define mechanisms of viral-mediated cardiac fibrosis.

PS-B01-7: VASCULAR SMOOTH MUSCLE CELL NOX5 REGULATES FIBROBLAST PHENOTYPIC SWITCH AND CARDIOVASCULAR FIBROSIS

Objective: We previously observed that mice expressing human Nox5 (hNox5) in VSMC exhibit vascular dysfunction and cardio-renal fibrosis, where mechanisms are unknown. Here we postulated that VSMC-Nox5 promotes fibroblast phenotypic changes leading to myofibroblast differentiation and pro-fibrotic responses. Design and Methods: Fibroblasts were cultured from wildtype (WT) and hNOX5 mice (Nox5 + SM22 +). Mice (20 weeks old) were infused with Ang II (600 ng/Kg/day) for 28 days and renal fibrosis/inflammation studied. Markers of myofibroblasts, pro-fibrotic and inflammatory phenotypes were assessed by qPCR and immunoblotting. Results: Fibroblasts from Nox5 + SM22 + mice exhibited increased mRNA of pro-fibrotic markers, such as Col1A1 (2-ddC:1.74 ± 0.16 vs. WT 0.67 ± 0.11), Col3A1 (2-ddC:1.74 ± 0.18 vs. WT 0.96 ± 0.24) and TIMP3 (2-ddC:2.65 ± 0.25 vs. WT 0.38 ± 0.07), p &lt; 0.05. Myofibroblast phenotype (SMC) markers mRNA, aSMA (2-ddC:1.54 ± 0.05 vs. WT 0.78 ± 0.17) and Myocd (2-ddC:1.36 ± 0.17 vs. WT 0.39 ± 0.22) were also increased, p &lt; 0.05. mRNA expression of CD36 (2-ddC:1.37 ± 0.07 vs. WT 86 ± 0.24), TNFa (2-ddC:1.32 ± 0.2 vs. WT 0.71 ± 0.17) and TNFR1 (2-ddC:1.26 ± 0.04 vs. WT 1.02 ± 0.10) were increased, while CD68 expression was decreased (2-ddC:0.82 ± 0.11 vs. WT 1.36 ± 0.18) in fibroblasts from Nox5+SM22+ mice (p &lt; 0.05). In Nox5 mice fibroblasts, ROS production and TGFb protein expression (AU:1.8 ± 0.05 vs. WT 1.4 ± 0.06), as well as TGFbR2 gene expression (2-ddC:2.04 ± 0.17 vs. WT 0.57 ± 0.12), were increased (p &lt; 0.05). Kidneys from Ang II-infused Nox5+SM22+ mice exhibited significant perivascular fibrosis and inflammatory cell infiltration compared to WT. Kidney expression of vimentin (AU:1.01 ± 0.05 vs WT 0.85 ± 0.03) and aSMA (AU:0.44 ± 0.03 vs WT 0.33 ± 0.01), markers of myofibroblast differentiation, were increased in Nox5 mice (p &lt; 0.05). Gene expression of DNMT3a and TET2, DNA methylation regulatory enzymes, were also increased in fibroblasts from Nox5+SM22+ mice, p &lt; 0.05. Conclusion: VSMC-Nox5 regulates fibroblast phenotypic switch leading to fibrosis; a process that may involve increased production of ROS and TGFb, and activation of DNA methylation pathways.

S-07-4: VASCULAR SMOOTH MUSCLE CELL PROTEOME AND PHENOTYPIC SWITCHING IN HUMAN HYPERTENSION ARE NOX5-DEPENDENT

Objective: Reactive oxygen species (ROS) play a key role in the regulation of vascular smooth muscle cell (VSMC) phenotypic switching in hypertension. Nox5 is a major ROS-generating enzyme in vascular cells and is upregulated in hypertension. However, the effects of Nox5-derived ROS on VSMC proteome and phenotype in human hypertension are unknown. We aimed to characterize the global and oxidative proteomic profile and VSMC phenotype in human hypertension and the role of Nox5. Design and method: VSMC from resistance arteries from normotensive (NT) and hypertensive (HT) subjects were studied (n = 5). Proteins were labelled with isobaric tandem mass tags and identified by liquid chromatography tandem mass spectrometry. The oxidative proteome was assessed using stable isotope-labelled iodoacetamide to target cysteine thiols. Nox5 silencing was performed by siRNA. Protein expression was detected by western blotting. The inflammatory, pro-fibrotic and mitogenic phenotype of VSMCs was assessed by measuring pro-inflammatory cytokines (IL-6, IL-8), pro-collagen I in the culture media. Results: Proteomic analysis identified 207 proteins upregulated in HT subjects (fold change &gt; 1.5, p &lt; 0.05). Gene ontology enrichment analysis of upregulated proteins in HT showed most proteins belong to extracellular space and plasma membrane compartments and were involved in extracellular matrix organization (ECM), immune response and cell proliferation. ECM proteins COL1A1, COL9A1, COL10A1, FBN1, FBLN1 were increased in cells from HT, suggesting a switch to a fibroblast-like phenotype in hypertension. Expression of proteins related to interferon and IL-1β pathways (IFIT1, IFIT2, IFIT3, MX1, MX2, ABCA1, ABCA2, IL1RAP, CD36, ICAM1) were also increased in cells from HT subjects. The VSMC oxidative proteome analysis identified 130 significantly regulated cysteine-containing peptides, 88 showed increased oxidation in HT (fold change&gt; 1.5, p &lt; 0.05). Among the highly oxidized proteins in HT were ECM proteins, COL11A1, COL16A1, FBLN1 and FBLN2. VSMCs from HT subjects exhibit increased expression of the proliferation marker, PCNA (0.162 ± 0.3 vs NT:0.051 ± 0.04 relative fluorescence units, p &lt; 0.05) and pro-collagen I (23.6 ± 2 vs NT:13.2 ± 0.3 ng/ml, p &lt; 0.05). Production of pro-inflammatory cytokines IL-6 (501.8 ± 23.6 vs NT:121.7 ± 6.4 pg/mL) and IL-8 (373.6 ± 34.1 vs NT:262.5 ± 24.6 pg/mL, p &lt; 0.05) were increased in HT. Nox5 silencing in VSMC from HT subjects reduced PCNA expression(43%), pro-collagen I release (8%), baseline and LPS-induced IL-6 (30% baseline, 43% LPS-induced) and IL-8 (21% baseline, 23% LPS-induced) release (p &lt; 0.05). Conclusions: Our study provides new insights into the proteomic changes related to vascular phenotype in hypertension and demonstrated that Nox5 plays an important role in VSMC phenotypic switching associated with vascular injury and remodelling in hypertension.

Autophagy in acute kidney injury and maladaptive kidney repair.

Acute kidney injury (AKI) is a major renal disease characterized by a sudden decrease in kidney function. After AKI, the kidney has the ability to repair, but if the initial injury is severe the repair may be incomplete or maladaptive and result in chronic kidney problems. Autophagy is a highly conserved pathway to deliver intracellular contents to lysosomes for degradation. Autophagy plays an important role in maintaining renal function and is involved in the pathogenesis of renal diseases. Autophagy is activated in various forms of AKI and acts as a defense mechanism against kidney cell injury and death. After AKI, autophagy is maintained at a relatively high level in kidney tubule cells during maladaptive kidney repair but the role of autophagy in maladaptive kidney repair has been controversial. Nonetheless, recent studies have demonstrated that autophagy may contribute to maladaptive kidney repair after AKI by inducing tubular degeneration and promoting a profibrotic phenotype in renal tubule cells. In this review, we analyze the role and regulation of autophagy in kidney injury and repair and discuss the therapeutic strategies by targeting autophagy.

Restoration of CPT1A-mediated fatty acid oxidation in mesothelial cells protects against peritoneal fibrosis.

Background: Peritoneal dialysis (PD) is limited by gradual fibrotic remodeling in the peritoneum, a process involving profibrotic response of mesothelial cells. However, the role of fatty acid oxidation (FAO) and carnitine palmitoyltransferase 1A (CPT1A) in this process remains unexplored. Methods: FAO and CPT1A expression were characterized in mesothelial cells from patients on long-term PD and from a mouse model of PD using multiple experimental methods, including single-cell sequencing, seahorse assay, real-time quantitative PCR, Western blot, and immunofluorescence staining. Overexpression of CPT1A was achieved in a human mesothelial cell line and in primary mouse mesothelial cells. Finally, genetic and pharmacological manipulations of CPT1A were performed in a mouse model of PD. Results: Herein, FAO and CPT1A expression were reduced in mesothelial cells from patients on long-term PD, which negatively correlated with expression of fibrogenic markers in these cells. This was corroborated in PD mice, as well as in mouse and human mesothelial cells incubated with transforming growth factor (TGF) β1. CPT1A overexpression in mesothelial cells, which prevented TGFβ1-induced suppression of mitochondrial respiration, restored cellular ATP levels and downregulated the expression of fibrogenic markers. Furthermore, restoration of FAO by overexpressing CPT1A in PD mice reversed profibrotic phenotype in mesothelial cells and reduced fibrotic lesions in the peritoneum. Treatment with the CPT1A activator C75 induced similar therapeutic benefit in PD mice. In contrast, inhibition of FAO with a CPT1 inhibitor caused more severe fibrosis in PD mice. Conclusions: A defective FAO is responsible for the profibrotic response of mesothelial cells and thus the peritoneal fibrogenesis. This aberrant metabolic state could be improved by modulating CPT1A in mesothelial cells, suggesting FAO enhancement in mesothelial cells is a potential treatment of peritoneal fibrosis.

An oral phenylacrylic acid derivative suppressed hepatic stellate cell activation and ameliorated liver fibrosis by blocking TGF-β1 signalling.

Liver fibrosis is an excessive wound-healing response governed by activated hepatic stellate cells (HSCs). To date, there is no drug available for liver fibrosis. Although ferulic acid (FA) has multiple pharmacological functions, its anti-hepatic fibrosis activity is weak. Based on the activity modification of the FA structure, we synthesized a series of phenylacrylic derivatives and found a superior compound, FA11. In this study, we investigated its antifibrotic effect and mechanism. Activated HSC and CCl4 -induced mouse liver fibrosis were established and followed by FA11 treatment. Cell viability was measured by CCK-8 assay. Apoptosis and cell cycle analysis were conducted by flow cytometry. Western blot and Real-time qPCR were used to examine the expression of fibrotic and M1/M2-type macrophages markers. Degree of liver fibrosis was shown by histological staining. In vitro, FA11 inhibited TGF-β1-induced LX-2 proliferation and led to apoptosis and cycle arrest. Furthermore, elevation of fibrotic markers in TGF-β1-induced LX-2 and primary activated HSC was reversed by FA11. In vivo, FA11 administration alleviated collagen deposition and blocked HSC activation and epithelial-mesenchymal transition (EMT). Additionally, FA11 reduced macrophage infiltration in fibrotic liver and prevented macrophage polarization to a profibrotic phenotype. Meanwhile, the systemic toxicity of CCl4 was also ameliorated by FA11. Mechanistically, FA11 reversed the phosphorylation of canonical and noncanonical TGF-β1 signalling, as well as FGFR1 signalling. We reported an oral phenylacrylic acid derivative, FA11, which showed excellent antifibrotic activity and was expected to be an anti-hepatic fibrosis candidate.

An alternative in vitro model considering cell-cell interactions in fiber-induced pulmonary fibrosis

Particularly since the wide-ranging health effects of asbestos exposure became known, great emphasis has been placed on detailed toxicity testing of known but also newly developed fiber materials. Exposure to respirable pollutants like fibers can lead to tissue injury causing lung diseases such as pulmonary fibrosis or cancer. In order to detect the toxic potential of such aerosols at an early stage, the development of suitable test systems is essential. In this study, we illustrate the development of an advanced in vitro cell model closely resembling the physiological structure of the alveoli, and we highlight its advantages over simpler models to predict pro-fibrotic changes. For this reason, we analyzed the cytotoxic effects of fiber-like multi-walled carbon nanotubes after 24 and 48 h exposure, and we investigated inflammatory, genotoxic and pro-fibrotic changes occurring in the developed triple culture consisting of lung epithelial cells, macrophages and fibroblasts compared to a co-culture of epithelial cells and fibroblasts or a mono culture of epithelial cells. In summary, the triple culture system is more precisely able to detect a pro-fibrotic phenotype including epithelial-mesenchymal transition as well as secondary genotoxicity, even if exhibiting lower cytotoxicity in contrast to the less advanced systems. These effects might be traced back to the complex interplay between the different cell types, all of which play an important role in the inflammatory response, which precedes wound healing, or even fibrosis or cancer development.

Epigenetic regulation of IPF fibroblast phenotype by glutaminolysis

ObjectiveExcessive extra-cellular-matrix production and uncontrolled proliferation of the fibroblasts are characteristics of many fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). The fibroblasts have enhanced glutaminolysis with up-regulated glutaminase, GLS1, which converts glutamine to glutamate. Here, we investigated the role of glutaminolysis and glutaminolysis-derived metabolite α-ketoglutarate (α-KG) on IPF fibroblast phenotype and gene expression. MethodsReduced glutamine conditions were carried out either using glutamine-free culture medium or silencing the expression of GLS1 with siRNA, with or without α-KG compensation. Cell phenotype has been characterized under these different conditions, and gene expression profile was examined by RNA-Seq. Specific profibrotic genes (Col3A1 and PLK1) expression were examined by real-time PCR and western blots. The levels of repressive histone H3K27me3, which demethylase activity is affected by glutaminolysis, were examined and H3K27me3 association with promoter region of Col3A1 and PLK1 were checked by ChIP assays. Effects of reduced glutaminolysis on fibrosis markers were checked in an animal model of lung fibrosis. ResultsThe lack of glutamine in the culture medium alters the profibrotic phenotype of activated fibroblasts. The addition of exogenous and glutaminolysis-derived metabolite α-KG to glutamine-free media barely restores the pro-fibrotic phenotype of activated fibroblasts. Many genes are down-regulated in glutamine-free medium, α-KG supplementation only rescues a limited number of genes. As α-KG is a cofactor for histone demethylases of H3K27me3, the reduced glutaminolysis alters H3K27me3 levels, and enriches H3K27me3 association with Col3A1 and PLK1 promoter region. Adding α-KG in glutamine-free medium depleted H3K27me3 association with Col3A1 promoter region but not that of PLK1. In a murine model of lung fibrosis, mice with reduced glutaminolysis showed markedly reduced fibrotic markers. ConclusionsThis study indicates that glutamine is critical for supporting pro-fibrotic fibroblast phenotype in lung fibrosis, partially through α-KG-dependent and –independent mechanisms, and supports targeting fibroblast metabolism as a therapeutic method for fibrotic diseases.

The Soluble Guanylate Cyclase Stimulator BAY 41-2272 Attenuates Transforming Growth Factor β1-Induced Myofibroblast Differentiation of Human Corneal Keratocytes

Corneal transparency, necessary for vision and depending on the high organization of stromal extracellular matrix, is maintained by keratocytes. Severe or continuous corneal injuries determine exaggerated healing responses resulting in the formation of irreversible fibrotic scars and vision impairment. Soluble guanylate cyclase (sGC) stimulation demonstrated antifibrotic effects in both experimental fibrosis and human lung and skin fibroblasts. Here, we assessed whether sGC stimulation with BAY 41-2272 could attenuate transforming growth factor β1 (TGFβ1)-induced myofibroblast differentiation of human corneal keratocytes. Cells were challenged with TGFβ1, with/without BAY 41-2272 preincubation, and subsequently assessed for viability, proliferation, migration, chemoinvasion, as well for the expression of myofibroblast/fibroblast activation markers and contractile abilities. Treatment with BAY 41-2272 did not affect keratocyte viability, while preincubation of cells with the sGC stimulator was able to inhibit TGFβ1-induced proliferation, wound healing capacity, and invasiveness. BAY 41-2272 was also able to attenuate TGFβ1-induced myofibroblast-like profibrotic phenotype of keratocytes, as demonstrated by the significant decrease in ACTA2, COL1A1, COL1A2, FN1 and PDPN gene expression, as well as in α-smooth muscle actin, α-1 chain of type I collagen, podoplanin, vimentin and N-cadherin protein expression. Finally, BAY 41-2272 significantly counteracted the TGFβ1-induced myofibroblast-like ability of keratocytes to contract collagen gels, reduced phosphorylated Smad3 protein levels, and attenuated gene expression of proinflammatory cytokines. Collectively, our data show for the first time that BAY 41-2272 is effective in counteracting keratocyte-to-myofibroblast transition, thus providing the rationale for the development of sGC stimulators as novel promising modulators of corneal scarring and fibrosis.

Mechanotransduction in the pathogenesis of non-alcoholic fatty liver disease.

Mechanobiology is a domain of interdisciplinary research that aims to explore the impact of physical force, applied externally or internally, on cell and tissue function, including development, growth, and differentiation. Mechanotransduction is a term that describes how cells sense physical forces (such as compression, stretch, and shear stress), convert them into biochemical signals, and mount adaptive responses integrated by the nucleus. There is accumulating evidence that mechanical forces extensively inform the biological behaviour of liver cells in health and disease. Recent research has elucidated many cellular and molecular mechanisms involved in this process including the pleiotropic control and diverse effects of the paralogous transcription co-activators YAP/TAZ, which play a prominent role in mechanotransduction. The liver sinusoids represent a unique microenvironment in which cells are exposed to mechanical cues originating in the cytoskeleton and at interfaces with adjacent cells, the extracellular matrix, and vascular or interstitial fluids. In non-alcoholic fatty liver disease (NAFLD), hepatocellular lipid accumulation and ballooning, activation of inflammatory responses, dysfunction of liver sinusoidal endothelial cells, and transdifferentiation of hepatic stellate cells into a pro-contractile and pro-fibrotic phenotype have been associated with aberrant cycles of mechanosensing and mechanoresponses. The downstream consequences of disrupted mechanical homeostasis likely contribute to the progression of NAFLD and promote the development of portal hypertension, cirrhosis, and hepatocellular carcinoma. Identification of molecular targets involved in pathogenic mechanotransduction will allow for the development of novel strategies to prevent the progression of liver disease in NAFLD.

Tacrolimus ameliorates bleomycin-induced pulmonary fibrosis by inhibiting M2 macrophage polarization via JAK2/STAT3 signaling.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease of unknown cause and characterized by excessive proliferation of fibroblasts and the irregular remodeling of extracellular matrix (ECM), which ultimately cause the severe distortion of the alveolar architecture. The median survival of IPF patients is 2-5years. IPF patients are predominantly infiltrated by M2 macrophages during the course of disease development and progression. Predominantly accumulation of M2 macrophages accelerates fibrosis progression by secreting multiple cytokines that promote fibroblast to myofibroblast transition. In the process of M2 macrophage polarization, JAK2/STAT3 signaling plays a key role, thus, targeting activated macrophages to inhibit the pro-fibrotic phenotype is considered as an approach to the potential treatment of IPF. Tacrolimus is a macrolide antibiotic that as a specific inhibitor of T-lymphocyte function and has been used widely as an immunosuppressant in human organ transplantation. In this study we explored the potential effect and mechanism of tacrolimus on pulmonary fibrosis in vivo and vitro. Here, we found that tacrolimus is capable of suppressing M2 macrophages polarization by inhibiting pro-fibrotic factors secreted by M2 macrophages. This effect further alleviates M2-induced myofibroblast activation, thus resulting in a decline of collagen deposition, pro-fibrotic cytokines secretion, recovering of lung function, ultimately relieving the progression of fibrosis in vivo. Mechanistically, we found that tacrolimus can inhibit the activation of JAK2/STAT3 signaling by targeting JAK2. Our findings indicate a potential anti-fibrotic effect of tacrolimus by regulating macrophage polarization and might be meaningful in clinical settings.

Extracellular Vesicles from Ocular Melanoma Have Pro-Fibrotic and Pro-Angiogenic Properties on the Tumor Microenvironment

Uveal melanoma (UM) is the most common primary intraocular tumor and often spreads to the liver. Intercellular communication though extracellular vesicles (EVs) plays an important role in several oncogenic processes, including metastasis, therapeutic resistance, and immune escape. This study examines how EVs released by UM cells modify stellate and endothelial cells in the tumor microenvironment. The surface markers, and the concentration and size of EVs derived from UM cells or choroidal melanocytes were characterized by high-resolution flow cytometry, electron microscopy, and Western blotting. The selective biodistribution of EVs was studied in mice by fluorescence imaging. The activation/contractility of stellate cells and the tubular organization of endothelial cells after exposure to melanomic EVs were determined by traction force microscopy, collagen gel contraction, or endothelial tube formation assays. We showed that large EVs from UM cells and healthy melanocytes are heterogenous in size, as well as their expression of phosphatidylserine, tetraspanins, and Tsg101. Melanomic EVs mainly accumulated in the liver and lungs of mice. Hepatic stellate cells with internalized melanomic EVs had increased contractility, whereas EV-treated endothelial cells developed more capillary-like networks. Our study demonstrates that the transfer of EVs from UM cells leads to a pro-fibrotic and pro-angiogenic phenotype in hepatic stellate and endothelial cells.

IL-10 is not anti-fibrotic but pro-fibrotic in endometriosis: IL-10 treatment of endometriotic stromal cells in vitro promotes myofibroblast proliferation and collagen type I protein expression.

Is interleukin-10 (IL-10) anti-fibrotic in endometriosis? IL-10 is not anti-fibrotic but pro-fibrotic in endometriosis, because IL-10 treatment of endometriotic stromal cells in vitro promotes myofibroblast proliferation and collagen type I protein expression. We previously showed that persistent activation of signal transducer and activator of transcription 3 (STAT3) via IL-6 trans-signaling promotes fibrosis of endometriosis. Studies showed marked anti-fibrotic effects of IL-10 via the STAT3 signaling pathway, which is generally considered to be anti-inflammatory, in various organs. Endometrial and/or endometriotic samples of 54 patients who had histological evidence of deep endometriosis, and endometrial samples from 30 healthy fertile women were analyzed. The effects of IL-10/STAT3 signaling as well as inhibition of STAT3 activation by knockdown of STAT3 gene on the pro-fibrotic phenotype in endometrial and endometriotic stromal cells in vitro were investigated. Then, the effects of various time points of IL-10 treatment in combination with transforming growth factor (TGF)-β1 and/or IL-6/soluble IL-6 receptor (sIL-6R) on the profibrotic phenotype of endometrial and endometriotic stromal cells were investigated. IL-10 induced pro-fibrotic phenotype (cell proliferation, collagen type I synthesis, α-smooth muscle actin positive stress fibers and collagen gel contraction) of endometriotic stromal cells. Knockdown of STAT3 gene decreased the IL-10 induced pro-fibrotic phenotype of endometriotic stromal cells. In contrast, IL-10 had no significant effects on pro-fibrotic phenotype of endometrial stromal cells of healthy women. Sequential IL-10 treatment with or without TGF-β1 and/or IL-6/sIL-6R induced persistent activation of STAT3 and significantly increased proliferation of myofibroblasts (cells with α-smooth muscle actin positive stress fibers) and protein expression of collagen type I in endometriotic stromal cells. TGF-β1 and/or IL-6/sIL6RIL-6/sIL6R treatment significantly increased tissue inhibitor of metalloproteinase 1 (TIMP1) protein expression, whereas IL-10 had no significant effects. Knockdown of STAT3 gene significantly decreased the TGF-β1 and/or IL-6/sIL6R induced TIMP1 protein expression. In contrast, pre-treatment with IL-10 before TGF-β1 and/or IL-6/sIL-6R treatment and sequential IL-10 treatment with or without TGF-β1 and/or IL-6/sIL-6R significantly decreased proliferation of fibroblasts (cells without α-smooth muscle actin positive stress fibers) and collagen type I protein expression in endometrial stromal cells of healthy women. N/A. Given the large number of complex interactions and signaling pathways of pro- and anti-inflammatory mediators that are involved in the pathophysiology of endometriosis, the present study investigated only a very small portion of the whole. Further in vivo studies are required to validate the present findings. Inflammatory mediators in the pathophysiology of endometriosis have been extensively investigated as potential therapeutic targets. However, the present study showed that anti-inflammatory signals of IL-10 and IL-6 through persistent STAT3 activation may promote endometriosis fibrosis. Therapeutic strategies, such as suppression of 'inflammation', might dysregulate the cross-regulation of 'pro- and anti-inflammatory mediators', leading to detrimental effects in patients with endometriosis, such as fibrosis. To develop new, but not deleterious, therapeutic strategies, studies are required to investigate whether, how and what 'anti-inflammatory mediators' along with pro-inflammatory mediators are involved in individual patients with endometriosis. This study was supported in part by KARL STORZ SE & Co. KG (Tuttlingen, Germany). The authors have no conflict of interest to disclose.