Role of Transglutaminase 2 in Cell Death, Survival, and Fibrosis.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme catalyzing the crosslinking between Gln and Lys residues and involved in various pathophysiological events. Besides this crosslinking activity, TG2 functions as a deamidase, GTPase, isopeptidase, adapter/scaffold, protein disulfide isomerase, and kinase. It also plays a role in the regulation of hypusination and serotonylation. Through these activities, TG2 is involved in cell growth, differentiation, cell death, inflammation, tissue repair, and fibrosis. Depending on the cell type and stimulus, TG2 changes its subcellular localization and biological activity, leading to cell death or survival. In normal unstressed cells, intracellular TG2 exhibits a GTP-bound closed conformation, exerting prosurvival functions. However, upon cell stimulation with Ca2+ or other factors, TG2 adopts a Ca2+-bound open conformation, demonstrating a transamidase activity involved in cell death or survival. These functional discrepancies of TG2 open form might be caused by its multifunctional nature, the existence of splicing variants, the cell type and stimulus, and the genetic backgrounds and variations of the mouse models used. TG2 is also involved in the phagocytosis of dead cells by macrophages and in fibrosis during tissue repair. Here, we summarize and discuss the multifunctional and controversial roles of TG2, focusing on cell death/survival and fibrosis.

Higher intestinal and circulatory lactate associated NOX2 activation leads to an ectopic fibrotic pathology following microcystin co-exposure in murine fatty liver disease.

Clinical studies implicated an increased risk of intestinal fibrosis in patients with nonalcoholic fatty liver disease (NAFLD). Our previous studies have shown that microcystin-LR (MC-LR) exposure led to altered gut microbiome and increased abundance of lactate producing bacteria and intestinal inflammation in underlying NAFLD. This led us to further investigate the effects of the MC-LR, a PP2A inhibitor in activating the TGF-ß fibrotic pathway in the intestines that might be mediated by increased lactate induced redox enzyme NOX2. Exposure to MC-LR led to higher lactate levels in circulation and in the intestinal content. The higher lactate levels were associated with NOX2 activation in vivo that led to increased Smad2/3-Smad4 co-localization and high alpha-smooth muscle actin (α-SMA) immunoreactivity in the intestines. Mechanistically, primary mouse intestinal epithelial cells treated with lactate and MC-LR separately led to higher NOX2 activation, phosphorylation of TGFßR1 receptor and subsequent Smad 2/3-Smad4 co-localization inhibitable by apocynin (NOX2 inhibitor), FBA (a peroxynitrite scavenger) and DMPO (a nitrone spin trap), catalase and superoxide dismutase. Inhibition of NOX2-induced redox signaling also showed a significant decrease in collagen protein thus suggesting a strong redox signaling induced activation of an ectopic fibrotic manifestation in the intestines. In conclusion, the present study provides mechanistic insight into the role of microcystin in dysbiosis-linked lactate production and subsequently advances our knowledge in lactate-induced NOX2 exacerbation of the cell differentiation and fibrosis in the NAFLD intestines.

Impact of Heparanse on Organ Fibrosis.

Organ fibrosis is defined as a deregulated wound-healing process characterized by a progressive accumulation of fibrous tissue and by reduced remodeling that can lead to the loss of functionality of the affected organ. This pathological process is quite common in several parenchymal organs such as kidneys, liver, and lungs and represents a real health emergency in developed western countries since a real anti-fibrotic therapy is not yet available in most cases. Heparanase (HPSE), which is the enzyme that cuts off the side chains of heparan sulfate (HS) proteoglycan, appears to be involved in the aetiopathogenesis of fibrosis in all these organs, even if with different mechanisms. Here we discuss how the interplay between HPSE and components of the immune and inflammatory responses can activate recruitment, proliferation, and activation of myofibroblasts which represent the main cell type responsible for the deposition of fibrous matrix. Finally, bearing in mind that once the activity of HPSE is inhibited no other molecule is able to perform the same function, it is desirable that this enzyme could prove to be a suitable pharmacological target in anti-fibrotic therapy.

Inhibition of glutaminase to reverse fibrosis in iatrogenic laryngotracheal stenosis.

OBJECTIVES/HYPOTHESIS: Glutamine metabolism is a critical energy source for iatrogenic laryngotracheal stenosis (iLTS) scar fibroblasts, and glutaminase (GLS) is an essential enzyme converting glutamine to glutamate. We hypothesize that the GLS-specific inhibitor BPTES will block glutaminolysis and reduce iLTS scar fibroblast proliferation, collagen deposition, and fibroblast metabolism in vitro. STUDY DESIGN: Test-tube Lab Research. METHODS: Immunohistochemistry of a cricotracheal resection (n = 1) and a normal airway specimen (n = 1) were assessed for GLS expression. GLS expression was assessed in brush biopsies of subglottic/tracheal fibrosis and normal airway from patients with iLTS (n = 6). Fibroblasts were isolated and cultured from biopsies of subglottic/tracheal fibrosis (n = 6). Fibroblast were treated with BPTES and BPTES + dimethyl α-ketoglutarate (DMK), an analogue of the downstream product of GLS. Fibroblast proliferation, gene expression, protein production, and metabolism were assessed in all treatment conditions and compared to control. RESULTS: GLS was overexpressed in brush biopsies of iLTS scar specimens (P = .029) compared to normal controls. In vitro, BPTES inhibited iLTS scar fibroblast proliferation (P = .007), collagen I (Col I) (P < .0001), collagen III (P = .004), and α-smooth muscle actin (P = .0025) gene expression and protein production (P = .031). Metabolic analysis demonstrated that BPTES reduced glycolytic reserve (P = .007) but had no effects on mitochondrial oxidative phosphorylation. DMK rescued BPTES inhibition of Col I gene expression (P = .0018) and protein production (P = .021). CONCLUSIONS: GLS is overexpressed in iLTS scar. Blockage of GLS with BPTES significantly inhibits iLTS scar fibroblasts proliferation and function, demonstrating a critical role for GLS in iLTS. Targeting GLS to inhibit glutaminolysis may be a successful strategy to reverse scar formation in the airway. LEVEL OF EVIDENCE: NA Laryngoscope, 2020.

Overexpression of MMPs in Corneas Requiring Penetrating and Deep Anterior Lamellar Keratoplasty.

Purpose: Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases involved in wound healing processes, including neovascularization and fibrosis. We assessed MMP protein expression levels in diseased corneas of patients requiring penetrating and deep anterior lamellar keratoplasty. The purpose of this study was to test the hypothesis that upregulation of MMPs in diseased corneas is positively associated with clinical levels of corneal neovascularization and fibrosis. Methods: Protein expression levels of nine individual MMPs were quantified simultaneously in human corneal lysates by using the Bio-Plex Pro Human MMP 9-Plex Panel and the MAGPIX technology. Measurements of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, and MMP13 were performed on diseased specimens from 21 patients undergoing corneal transplantation (17 for penetrating keratoplasty and 4 for deep anterior lamellar keratoplasty) and 6 normal control corneas. Results: Luminex-based expression analysis revealed a significant overexpression of four of the nine MMPs tested (MMP2, MMP8, MMP12, and MMP13) in patient samples compared to control. Significant overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 was observed in diseased corneas with neovascularization compared with diseased corneas without neovascularization. Overexpression of MMP1, MMP2, MMP8, MMP12, and MMP13 also corresponded with the levels of corneal fibrosis. Finally, reduced expression of MMP3 was detected in keratoconus patients. Conclusions: Multiple MMPs are expressed in the corneas of patients with chronic disease requiring keratoplasty even when the pathologic process appears to be clinically inactive. In particular, the expression of several MMPs (MMP2, MMP8, MMP12, and MMP13) is positively associated with increased levels corneal fibrosis and neovascularization.

Decreased catalase expression is associated with ligamentum flavum hypertrophy due to lumbar spinal canal stenosis.

BACKGROUND: This is an immunohistologic study of gene expression between patients and controls.This study aims to evaluate expression of the catalase gene in hypertrophied ligamentum flavum (LF) specimens obtained from patients with lumbar spinal canal stenosis (LSCS).LSCS is one of the most common spinal disorders. It is well known that LF hypertrophy plays an important role in the onset of LSCS. Although degenerative changes, aging, and mechanical stress are all thought to contribute to hypertrophy and fibrosis of the LF, the precise pathogenesis of LF hypertrophy remains unknown. Previous genetic studies have tried to determine the mechanism of LF hypertrophy. However, the association between catalase gene expression and LF hypertrophy has not yet been explored. METHODS: LF specimens were surgically obtained from 30 patients with spinal stenosis (LSCS group) and from 30 controls with lumbar disc herniation (LDH group). LF thickness was measured at the thickest point using calipers to an accuracy of 0.01 mm during surgical intervention. The extent of LF elastin degradation and fibrosis were graded (grades 0-4) by hematoxylin and eosin staining and Masson trichrome staining, respectively. The resulting LF measurements, histologic data, and immunohistologic results were then compared between the 2 groups. RESULTS: The average LF thickness was significantly higher in the LSCS group than in the LDH group (5.99 and 2.95 mm, respectively, P = .004). Elastin degradation and fibrosis of the LF were significantly more severe in spinal stenosis samples than in the disc herniation samples (3.04 ±â€Š0.50 vs 0.79 ±â€Š0.60, P = .007; 3.01 ±â€Š0.47 vs 0.66 ±â€Š0.42, P = .009, respectively). Significantly lower expression of catalase was observed in the perivascular area of LF samples obtained from patients with LSCS compared with controls (61.80 ±â€Š31.10 vs 152.80 ±â€Š41.13, respectively, P = .009). CONCLUSION: Our findings suggest that decreased expression of catalase is associated with LF hypertrophy in patients with LSCS.

First description of a compensatory xylosyltransferase I induction observed after an antifibrotic UDP-treatment of normal human dermal fibroblasts.

Fibrosis is a serious health problem often leading to accompanying organ failure. During the manifestation of the disease, an accumulation of different extracellular matrix (ECM) molecules, such as proteoglycans, takes place. There is no appropriate therapeutic option available to heal fibrosis to date. Current research focuses primarily on targets such as the cytokine transforming growth factor-ß1 (TGF-ß1), which is assumed to be one of the key mediators of fibrosis. Both xylosyltransferase isoforms, XT-I and XT-II, catalyze the rate-limiting step of the proteoglycan biosynthesis. Consequently, inhibiting XT activity could be a promising approach to treat fibrosis. It was shown in earlier studies that nucleotides and nucleosides have anti-fibrotic properties and decrease XT activity in cell-free systems. In contrast, we evaluated the mechanisms beyond an UDP-mediated induction of intracellular XT-activity in normal human dermal fibroblasts (NHDF). The observed pseudo-fibrotic XT increasement could be attributed to a compensation of decreased UDP-glucuronate decarboxylase 1 (UXS1) mRNA expression as well as a diminished intracellular UDP-xylose concentration. In summary, our results describe a so far unknown XT-inductive pathway and show that UDP could be a promising molecule for the development of an anti-fibrotic therapy. Nevertheless, XT activity has to be inhibited in parallel intracellularly.

The lysyl oxidase like 2/3 enzymatic inhibitor, PXS-5153A, reduces crosslinks and ameliorates fibrosis.

Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε-amino group of lysine or hydroxylysine on collagen side-chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS-5153A, a novel mechanism based, fast-acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS-5153A dose-dependently reduced LOXL2-mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet-induced, PXS-5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS-5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.

Quercetin Shows the Pharmacological Activity to Simultaneously Downregulate the Inflammatory and Fibrotic Responses to Tissue Injury in Association with its Ability to Target Multi-Kinases.

AIM: Previous studies have suggested that quercetin is effective for treating diverse chronic disorders including organ fibrosis and airway and cardiovascular disorders. To access the pharmacological background for its broad efficacy, we examined the ability of quercetin to modulate the inflammatory and fibrotic responses associated with organ injury that commonly underlie the pathogenesis of those disorders. METHODS: A cutaneous wound model on rabbit ear was used for in vivo study. Quercetin was topically applied to the wounds, and the number of macrophages and myofibroblasts and the size of the hypertrophic scar formed were estimated. An in vitro study examined the ability of quercetin to inhibit cell-signaling pathways that activate RAW264.7 macrophages and primary dermal fibroblasts and the tyrosine kinase activity of discoidin domain receptor 2. RESULTS: Quercetin reduced the population of macrophages and myofibroblasts and the scar formation in cutaneous wound healing. Quercetin suppressed the signaling pathways activating RAW264.7 macrophages and dermal fibroblasts, which is associated with its inhibition of multiple tyrosine kinases to regulate the pathways. This pharmacological activity of quercetin to simultaneously inhibit the inflammatory and fibrotic responses upon tissue damage by targeting multi-kinases could be the action mechanism to support its broad efficacy for various chronic disorders.

PARP-1 inhibition attenuates cardiac fibrosis induced by myocardial infarction through regulating autophagy.

Post-MI heart failure is characterized by structural remodeling, in which intramyocardial fibrosis takes a important part. Poly(ADP-ribose) polymerase 1 (PARP-1) is a extensive nuclear enzyme and plays a critical role in various diseases. It was shown that PARP-1 inhibition could alleviate heart failure and dowregulate autophagy, but whether PARP-1 regulates autophagy and thus impacts the activities of CFs remain unknown. We transfected cultured cardiac fibroblasts (CFs) with small interfere RNA-PARP-1 (siPARP-1) to downregulate PARP-1 and analyzed the ability of proliferation, migration, differentiation, and autophagy levels of CFs under different treatments using CCK8 assays, transwell migration assays, immunofluorescence assays detecting expression of α-SMA, western blot assays detecting autophagy-related proteins respectively. Furthermore, rat models of myocardial infarction (MI) were induced by ligation of left anterior descending coronary artery and PARP-1 inhibitor, 4-aminobenzamide (4-AB), was injected intraperitoneally after MI, followed by echocardiography detection, masson assays, immunohistochemistry assays detecting expression of α-SMA and western blot assays detecting autophagy-related proteins to investigate whether PARP-1 inhibition could regulate autophagy, alleviate cardiac fibrosis and improve cardiac function in vivo. In cultured CFs, siPARP-1 repressed TGF-ß1-induced proliferation, migration, and differentiation through regulating autophagic levels. The in vitro results was verified by the in vivo study, indicating that PARP-1 inhibition partially decreased autophagy, abrogated cardiac fibrosis and significantly improved cardiac function post-MI. In conclusion, this work demonstrated the vital connection of PARP-1 and autophagy in the activation of CFs, and provided solid evidence supporting PARP-1 inhibition as a feasible strategy for the treatment of post-MI heart failure.

Pharmacological Inhibition of Soluble Epoxide Hydrolase Ameliorates Chronic Ethanol-Induced Cardiac Fibrosis by Restoring Autophagic Flux.

BACKGROUND: Chronic drinking leads to myocardial contractile dysfunction and dilated cardiomyopathy, and cardiac fibrosis is a consequence of these alcoholic injuries. Soluble epoxide hydrolase (sEH) hydrolyzes epoxyeicosatrienoic acids (EETs) to less bioactive diols, and EETs have cardioprotective properties. However, the effects of sEH inhibition in ethanol (EtOH)-induced cardiac fibrosis are unknown. METHODS: This study was designed to investigate the role and underlying mechanisms of sEH inhibition in chronic EtOH feeding-induced cardiac fibrosis. C57BL/6J mice were fed a 4% Lieber-DeCarli EtOH diet for 8 weeks, and the sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered throughout the experimental period. RESULTS: The results showed that chronic EtOH intake led to cardiac dilatation, collagen deposition, and autophagosome accumulation, while TPPU administration ameliorated these effects. In vitro, treating primary cardiac fibroblasts (CFs) with EtOH resulted in CF activation, including alpha smooth muscle actin overexpression, collagen synthesis, and cell migration. Moreover, EtOH disturbed CF autophagic flux, as evidenced by the increased LC3 II/I ratio and SQSTM1 expression, and by the enhanced autophagosome accumulation. TPPU treatment prevented the activation of CF induced by EtOH and restored the impaired autophagic flux by suppressing mTOR activation. CONCLUSIONS: Taken together, these findings suggest that sEH pharmacological inhibition may be a unique therapeutic strategy for treating EtOH-induced cardiac fibrosis.

C1QTNF1 attenuates angiotensin II-induced cardiac hypertrophy via activation of the AMPKa pathway.

RATIONALE: Complement C1q tumor necrosis factor related proteins (C1QTNFs) have been reported to have diverse biological influence on the cardiovascular system. C1QTNF1 is a member of the CTRP superfamily. C1QTNF1 is expressed in the myocardium; however, its function in myocytes has not yet been investigated. OBJECTIVE: To systematically investigate the roles of C1QTNF1 in angiotensin II (Ang II)-induced cardiac hypertrophy. METHODS AND RESULTS: C1QTNF1 knock-out mice were used with the aim of determining the role of C1QTNF1 in cardiac hypertrophy in the adult heart. Data from experiments showed that C1QTNF1 was up-regulated during cardiac hypertrophic processes, which were triggered by increased reactive oxygen species. C1QTNF1 deficiency accelerated cardiac hypertrophy, fibrosis, inflammation responses, and oxidative stress with deteriorating cardiac dysfunction in the Ang II-induced cardiac hypertrophy mouse model. We identified C1QTNF1 as a negative regulator of cardiomyocyte hypertrophy in Ang II-stimulated neonatal rat cardiomyocytes using the recombinant human globular domain of C1QTNF1 and C1QTNF1 siRNA. Injection of the recombinant human globular domain of C1QTNF1 also suppressed the Ang II-induced cardiac hypertrophic response in vivo. The anti-hypertrophic effects of C1QTNF1 rely on AMPKa activation, which inhibits mTOR P70S6K phosphorylation. An AMPKa inhibitor abrogated the anti-hypertrophic effects of the recombinant human globular domain of C1QTNF1 both in vivo and vitro. Moreover, C1QTNF1-mediated AMPKa activation was triggered by the inhibition of PDE1-4, which subsequently activated the cAMP/PKA/LKB1 pathway. CONCLUSION: Our results demonstrated that C1QTNF1 improves cardiac function and inhibits cardiac hypertrophy and fibrosis by increasing and activating AMPKa, suggesting that C1QTNF1 could be a therapeutic target for cardiac hypertrophy and heart failure.

Poly(ADP-ribose) polymerase-1 regulates fibroblast activation in systemic sclerosis.

OBJECTIVES: The enzyme poly(ADP-ribose) polymerase-1 (PARP-1) transfers negatively charged ADP-ribose units to target proteins. This modification can have pronounced regulatory effects on target proteins. Recent studies showed that PARP-1 can poly(ADP-ribosyl)ate (PARylate) Smad proteins. However, the role of PARP-1 in the pathogenesis of systemic sclerosis (SSc) has not been investigated. METHODS: The expression of PARP-1 was determined by quantitative PCR and immunohistochemistry. DNA methylation was analysed by methylated DNA immunoprecipitation assays. Transforming growth factor-ß (TGFß) signalling was assessed using reporter assays, chromatin immunoprecipitation assays and target gene analysis. The effect of PARP-1 inactivation was investigated in bleomycin-induced and topoisomerase-induced fibrosis as well as in tight-skin-1 (Tsk-1) mice. RESULTS: The expression of PARP-1 was decreased in patients with SSc, particularly in fibroblasts. The promoter of PARP-1 was hypermethylated in SSc fibroblasts and in TGFß-stimulated normal fibroblasts. Inhibition of DNA methyltransferases (DNMTs) reduced the promoter methylation and reactivated the expression of PARP-1. Inactivation of PARP-1 promoted accumulation of phosphorylated Smad3, enhanced Smad-dependent transcription and upregulated the expression of TGFß/Smad target genes. Inhibition of PARP-1 enhanced the effect of TGFß on collagen release and myofibroblast differentiation in vitro and exacerbated experimental fibrosis in vivo. PARP-1 deficiency induced a more severe fibrotic response to bleomycin with increased dermal thickening, hydroxyproline content and myofibroblast counts. Inhibition of PARylation also exacerbated fibrosis in Tsk-1 mice and in mice with topoisomerase-induced fibrosis. CONCLUSION: PARP-1 negatively regulates canonical TGFß signalling in experimental skin fibrosis. The downregulation of PARP-1 in SSc fibroblasts may thus directly contribute to hyperactive TGFß signalling and to persistent fibroblast activation in SSc.

Role of the Autotaxin-LPA Pathway in Dexamethasone-Induced Fibrotic Responses and Extracellular Matrix Production in Human Trabecular Meshwork Cells.

Purpose: Dexamethasone (Dex) regulates aqueous humor outflow by inducing reorganization of the cytoskeleton and extracellular matrix (ECM) production. Rho kinase (ROCK) has an important role in this process, but the upstream pathway leading to its activation remains elusive. The purpose of the study was to determine the role of autotaxin (ATX), an enzyme involved in the generation of lysophosphatidic acid (LPA), in the Dex-induced fibrotic response and ECM production in human trabecular meshwork (HTM) cells. Methods: The expression of ATX in specimens from glaucoma patients was investigated by immunohistochemistry. Regulation of ATX expression and the changes in actin cytoskeleton, ECM production, myosin light chain (MLC) and cofilin phosphorylation, ATX secretion, and lysophospholipase D (lysoPLD) activity induced by Dex treatment in HTM cells were determined by immunofluorescence, real-time quantitative PCR, immunoblot, and the two-site immunoenzymetric and lysoPLD assays. Results: Significant ATX expression was found in conventional outflow pathway specimens from glaucoma patients. Dex treatment induced increases in ATX mRNA levels, protein expression, and secretion in HTM cells in association with reorganization of cytoskeleton and ECM accumulation. Significant suppression of these aforementioned changes was observed after ATX/LPA-receptor/ROCK inhibition as well as suppression of fibrotic changes and MLC and cofilin phosphorylation in HTM cells. Conclusions: The results of this study, including the robust induction of ATX by Dex treatment, in association with fibrotic changes and ECM production in HTM cells, collectively suggest a potential role for ATX-LPA pathway in the regulation of aqueous humor outflow and IOP in glaucomatous eyes.

Copper-induced reduction in myocardial fibrosis is associated with increased matrix metalloproteins in a rat model of cardiac hypertrophy.

Trientine (TETA), a copper (Cu) chelator, is capable of replenishing Cu in the heart, and Cu repletion reduces cardiac fibrosis in a rodent model of cardiac hypertrophy. This study was undertaken to explore possible mechanisms by which Cu repletion diminishes cardiac fibrosis. Adult male Sprague-Dawley rats were subjected to ascending aortic constriction to induce cardiac hypertrophy. Four months after the operation, cardiac hypertrophy along with fibrosis was fully developed. TETA treatment was then followed at a dose of 21.9 mg kg-1, twice a day, administered orally for six weeks. At the end of the treatment, the hearts were harvested and all of the tissue samples were subjected to qRT-PCR, western blot, Sirius red staining, hydroxyproline assay, and immunostaining analyses. TETA treatment significantly increased the content of Cu in the hypertrophied myocardium, decreased type III collagen deposition and reduced cardiac fibrosis. On the other hand, this treatment did not alter the increase in fibroblasts induced by pressure overload, but significantly increased matrix metalloproteinase-2 (MMP-2), which is the enzyme mainly responsible for degradation of collagens in the heart. In addition, the mRNA and protein levels of tissue inhibitors of matrix metalloproteinase-1 and -2 (TIMP-1 and TIMP-2) were both remarkably increased in the hypertrophic myocardium, and normalized after TETA treatment. The data thus demonstrated that the reduction in cardiac fibrosis by TETA-induced Cu repletion is associated at least in part with an enhanced MMP-2 activity, leading to collagen degradation.

The histone demethylase Jumonji domain-containing protein 3 (JMJD3) regulates fibroblast activation in systemic sclerosis.

OBJECTIVES: Systemic sclerosis (SSc) fibroblasts remain activated even in the absence of exogenous stimuli. Epigenetic alterations are thought to play a role for this endogenous activation. Trimethylation of histone H3 on lysine 27 (H3K27me3) is regulated by Jumonji domain-containing protein 3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) in a therapeutically targetable manner. The aim of this study was to explore H3K27me3 demethylases as potential targets for the treatment of fibrosis. METHODS: JMJD3 was inactivated by small interfering RNA-mediated knockdown and by pharmacological inhibition with GSKJ4. The effects of targeted inactivation of JMJD3 were analysed in cultured fibroblasts and in the murine models of bleomycin-induced and topoisomerase-I (topoI)-induced fibrosis. H3K27me3 at the FRA2 promoter was analysed by ChIP. RESULTS: The expression of JMJD3, but not of UTX, was increased in fibroblasts in SSc skin and in experimental fibrosis in a transforming growth factor beta (TGFß)-dependent manner. Inactivation of JMJD3 reversed the activated fibroblast phenotype in SSc fibroblasts and prevented the activation of healthy dermal fibroblasts by TGFß. Pharmacological inhibition of JMJD3 ameliorated bleomycin-induced and topoI-induced fibrosis in well-tolerated doses. JMJD3 regulated fibroblast activation in a FRA2-dependent manner: Inactivation of JMJD3 reduced the expression of FRA2 by inducing accumulation of H3K27me3 at the FRA2 promoter. Moreover, the antifibrotic effects of JMJD3 inhibition were reduced on knockdown of FRA2. CONCLUSION: We present first evidence for a deregulation of JMJD3 in SSc. JMJD3 modulates fibroblast activation by regulating the levels of H3K27me3 at the promoter of FRA2. Targeted inhibition of JMJD3 limits the aberrant activation of SSc fibroblasts and exerts antifibrotic effects in two murine models.

Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP).

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

Serum Matrix Metalloproteinase-7 Level is Associated with Fibrosis and Renal Survival in Patients with IgA Nephropathy.

BACKGROUND/AIMS: In view of the latest findings that matrix metalloproteinase-7 (MMP-7) acted as a vital marker and pathogenic mediator of renal fibrosis in a murine model, we hypothesized that serum MMP-7 level might serve as a noninvasive prognostic biomarker in IgA nephropathy (IgAN) patients. METHODS: We conducted a retrospective follow-up study of 244 IgAN patients for a median of 81.9 months. Serum MMP-7 was detected at the time of diagnosis, and renal progression was assessed by Cox proportional hazards method. RESULTS: Compared with healthy populations, the serum levels of MMP-7 were significantly elevated in IgAN patients. Besides, serum MMP-7 levels were well correlated with renal scarring lesions characterized by glomerular sclerosis and interstitial fibrosis. Follow-up analyses revealed that increased serum MMP-7 levels were linked with a greater risk of poor renal outcome with a hazard ratio of 1.898 per doubling MMP-7 concentration. By contrast with the first quartile, the risk of deterioration in renal function elevated such that the hazard ratio for the second quartile was 1.805, 3.383 for the third, and 5.173 for the fourth quartile of the MMP-7 level. CONCLUSIONS: This study showed that the higher serum MMP-7 levels were independently associated with renal fibrosis and poor prognosis in IgAN.

BMP7-induced-Pten inhibits Akt and prevents renal fibrosis.

Bone morphogenetic protein-7 (BMP-7) counteracts pro-fibrotic effects of TGFß1 in cultured renal cells and protects from fibrosis in acute and chronic renal injury models. Using the unilateral ureteral obstruction (UUO) model of chronic renal fibrosis, we investigated the effect of exogenous-rhBMP-7 on pro-fibrotic signaling pathways mediated by TGFß1 and hypoxia. Mice undergoing UUO were treated with vehicle or rhBMP-7 (300µg/kg i.p.) every other day for eight days and kidneys analysed for markers of fibrosis and SMAD, MAPK, and PI3K signaling. In the kidney, collecting duct and tubular epithelial cells respond to BMP-7 via activation of SMAD1/5/8. Phosphorylation of SMAD1/5/8 was reduced in UUO kidneys from vehicle-treated animals yet maintained in UUO kidneys from BMP-7-treated animals, confirming renal bioactivity of exogenous rhBMP-7. BMP-7 inhibited Collagen Iα1 and Collagen IIIα1 gene expression and Collagen I protein accumulation, while increasing expression of Collagen IVα1 in UUO kidneys. Activation of SMAD2, SMAD3, ERK, p38 and PI3K/Akt signaling occurred during fibrogenesis and BMP-7 significantly attenuated SMAD3 and Akt signaling in vivo. Analysis of renal collecting duct (mIMCD) and tubular epithelial (HK-2) cells stimulated with TGFß1 or hypoxia (1% oxygen) to activate Akt provided further evidence that BMP-7 specifically inhibited PI3K/Akt signaling. PTEN is a negative regulator of PI3K and BMP-7 increased PTEN expression in vivo and in vitro. These data demonstrate an important mechanism by which BMP-7 orchestrates renal protection through Akt inhibition and highlights Akt inhibitors as anti-fibrotic therapeutics.

Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available. Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study, we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced the mRNA level of profibrotic genes, including collagen 1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single-treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator-activated receptor γ coactivator-1α mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.-Nio, Y., Tanaka, M., Hirozane, Y., Muraki, Y., Okawara, M., Hazama, M., Matsuo, T. Phosphodiesterase 4 inhibitor and phosphodiesterase 5 inhibitor combination therapy has antifibrotic and anti-inflammatory effects in mdx mice with Duchenne muscular dystrophy.