Novel Omega-3 Fatty Acid Epoxygenase Metabolite Reduces Kidney Fibrosis

Cytochrome P450 (CYP) monooxygenases epoxidize the omega-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid into novel epoxydocosapentaenoic acids (EDPs) that have multiple biological actions. The present study determined the ability of the most abundant EDP regioisomer, 19,20-EDP to reduce kidney injury in an experimental unilateral ureteral obstruction (UUO) renal fibrosis mouse model. Mice with UUO developed kidney tubular injury and interstitial fibrosis. UUO mice had elevated kidney hydroxyproline content and five-times greater collagen positive fibrotic area than sham control mice. 19,20-EDP treatment to UUO mice for 10 days reduced renal fibrosis with a 40%–50% reduction in collagen positive area and hydroxyproline content. There was a six-fold increase in kidney α-smooth muscle actin (α-SMA) positive area in UUO mice compared to sham control mice, and 19,20-EDP treatment to UUO mice decreased α-SMA immunopositive area by 60%. UUO mice demonstrated renal epithelial-to-mesenchymal transition (EMT) with reduced expression of the epithelial marker E-cadherin and elevated expression of multiple mesenchymal markers (FSP-1, α-SMA, and desmin). Interestingly, 19,20-EDP treatment reduced renal EMT in UUO by decreasing mesenchymal and increasing epithelial marker expression. Overall, we demonstrate that a novel omega-3 fatty acid metabolite 19,20-EDP, prevents UUO-induced renal fibrosis in mice by reducing renal EMT.     

Paricalcitol Improves Hypoxia-Induced and TGF-β1-Induced Injury in Kidney Pericytes

Recently, the role of kidney pericytes in kidney fibrosis has been investigated. This study aims to evaluate the effect of paricalcitol on hypoxia-induced and TGF-β1-induced injury in kidney pericytes. The primary cultured pericytes were pretreated with paricalcitol (20 ng/mL) for 90 min before inducing injury, and then they were exposed to TGF-β1 (5 ng/mL) or hypoxia (1% O₂ and 5% CO₂). TGF-β1 increased α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericytes, whereas paricalcitol reversed the changes. Paricalcitol inhibited the TGF-β1-induced cell migration of pericytes. Hypoxia increased TGF-β1, α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericyte, whereas paricalcitol reversed them. Hypoxia activated the HIF-1α and downstream molecules including prolyl hydroxylase 3 and glucose transporter-1, whereas paricalcitol attenuated the activation of the HIF-1α-dependent molecules and TGF-β1/Smad signaling pathways in hypoxic pericytes. The gene silencing of HIF-1α vanished the hypoxia-induced TGF-β1, α-SMA upregulation, and PDGFRβ downregulation. The effect of paricalcitol on the HIF-1α-dependent changes of fibrosis markers was not significant after the gene silencing of HIF-1α. In addition, hypoxia aggravated the oxidative stress in pericytes, whereas paricalcitol reversed the oxidative stress by increasing the antioxidant enzymes in an HIF-1α-independent manner. In conclusion, paricalcitol improved the phenotype changes of pericyte to myofibroblast in TGF-β1-stimulated pericytes. In addition, paricalcitol improved the expression of fibrosis markers in hypoxia-exposed pericytes both in an HIF-1α-dependent and independent manner.     

Neoagarooligosaccharide Protects against Hepatic Fibrosis via Inhibition of TGF-β/Smad Signaling Pathway

Hepatic fibrosis occurs when liver tissue becomes scarred from repetitive liver injury and inflammatory responses; it can progress to cirrhosis and eventually to hepatocellular carcinoma. Previously, we reported that neoagarooligosaccharides (NAOs), produced by the hydrolysis of agar by β-agarases, have hepatoprotective effects against acetaminophen overdose-induced acute liver injury. However, the effect of NAOs on chronic liver injury, including hepatic fibrosis, has not yet been elucidated. Therefore, we examined whether NAOs protect against fibrogenesis in vitro and in vivo. NAOs ameliorated PAI-1, α-SMA, CTGF and fibronectin protein expression and decreased mRNA levels of fibrogenic genes in TGF-β-treated LX-2 cells. Furthermore, downstream of TGF-β, the Smad signaling pathway was inhibited by NAOs in LX-2 cells. Treatment with NAOs diminished the severity of hepatic injury, as evidenced by reduction in serum alanine aminotransferase and aspartate aminotransferase levels, in carbon tetrachloride (CCl₄)-induced liver fibrosis mouse models. Moreover, NAOs markedly blocked histopathological changes and collagen accumulation, as shown by H&E and Sirius red staining, respectively. Finally, NAOs antagonized the CCl₄-induced upregulation of the protein and mRNA levels of fibrogenic genes in the liver. In conclusion, our findings suggest that NAOs may be a promising candidate for the prevention and treatment of chronic liver injury via inhibition of the TGF-β/Smad signaling pathway.     

Effect of Hypoxia on Pulmonary Endothelial Cells from Bleomycin-Induced Pulmonary Fibrosis Model Mice

Pulmonary fibrosis is a progressive and fatal disorder characterized by dysregulated repair after recurrent injury. Destruction of the lung architecture with excess extracellular matrix deposition induces respiratory failure with hypoxia and progressive dyspnea. The impact of hypoxia on pulmonary endothelial cells during pulmonary fibrogenesis is unclear. Using a magnetic-activated cell sorting system, pulmonary endothelial cells were isolated from a mouse model of pulmonary fibrosis induced by intratracheally administered bleomycin. When endothelial cells were exposed to hypoxic conditions, a hypoxia-inducible factor (HIF)-2α protein was detected in CD31- and α-smooth muscle actin (SMA)-positive cells. Levels of plasminogen activator inhibitor 1, von Willebrand factor, and matrix metalloproteinase 12 were increased in endothelial cells isolated from bleomycin-treated mice exposed to hypoxic conditions. When endothelial cells were cultured under hypoxic conditions, levels of fibrotic mediators, transforming growth factor-β and connective tissue growth factor, were elevated only in endothelial cells from bleomycin-treated and not from saline-treated lungs. The increased expression of α-SMA and mesenchymal markers and collagen production in bleomycin- or hypoxia-stimulated endothelial cells were further elevated in endothelial cells from bleomycin-treated mouse lungs cultured under hypoxic conditions. Exposure to hypoxia damaged endothelial cells and enhanced fibrogenesis-related damage in bleomycin-treated pulmonary endothelial cells.     

Fibrotic Changes to Schlemm’s Canal Endothelial Cells in Glaucoma

Previous studies have shown that glaucomatous Schlemm’s canal endothelial cells (gSCECs) are stiffer and associated with reduced porosity and increased extracellular matrix (ECM) material compared to SCECs from healthy individuals. We hypothesised that Schlemm’s canal (SC) cell stiffening was a function of fibrotic changes occurring at the inner wall of SC in glaucoma. This study was performed in primary cell cultures isolated from the SC lumen of human donor eyes. RNA and protein quantification of both fibrotic and endothelial cell markers was carried out on both healthy and gSCECs. Functional assays to assess cell density, size, migration, proliferation, and mitochondrial function of these cells were also carried out. Indeed, we found that gSCECs deviate from typical endothelial cell characteristics and exhibit a more fibrotic phenotype. For example, gSCECs expressed significantly higher protein levels of the fibrotic markers α-SMA, collagen I-α1, and fibronectin, as well as significantly increased protein expression of TGFβ-2, the main driver of fibrosis, compared to healthy SCECs. Interestingly, we observed a significant increase in protein expression of endothelial marker VE-cadherin in gSCECs, compared to healthy SCECs. gSCECs also appeared to be significantly larger, and surprisingly proliferate and migrate at a significantly higher rate, as well as showing significantly reduced mitochondrial activity, compared to healthy SCECs.     

Adenosine A3 Receptor (A3AR) Agonist for the Treatment of Bleomycin-Induced Lung Fibrosis in Mice

Adenosine receptors (ARs) are involved in the suppression and development of inflammatory and fibrotic conditions. Specifically, AR activation promotes differentiation of lung fibroblasts into myofibroblasts, typical of a fibrotic event. Pulmonary fibrosis is a severe disease characterized by inflammation and fibrosis of unknown etiology and lacking an effective treatment. The present investigation explored the action of MRS5980, a new, highly potent and selective A₃AR agonist, in an established murine model of lung fibrosis. The effects of either vehicle or MRS5980 were studied in mice following intratracheal bleomycin administration. We evaluated the role of the A₃AR agonist on lung stiffness, studying the airway resistance to inflation, oxidative stress (8-OHdG and MDA), inflammation, pro- and anti-inflammatory marker levels (IL-1β, IL-6, TNF-α, IL-10 and IL-17A) and fibrosis establishment, evaluating transforming growth factor (TGF)-β expression and α-smooth muscle actin (α-SMA) deposition in lungs. Bleomycin administration increased lung stiffness, TGF-β levels, α-SMA deposition, and inflammatory and oxidative stress markers. The treatment with MRS5980 attenuated all the analyzed functional, biochemical and histopathological markers in a dose-dependent manner. Our findings support the therapeutic potential of A₃AR agonists in lung fibrosis by demonstrating reduced disease progression, as indicated by decreased inflammation, TGF-β expression and fibrotic remodeling.     

Ghrelin Attenuates Liver Fibrosis through Regulation of TGF-β1 Expression and Autophagy

Ghrelin is a stomach-derived growth hormone secretagogue that promotes various physiological effects, including energy metabolism and amelioration of inflammation. The purpose of this study was to investigate the protective mechanism of ghrelin against liver fibrosis. Liver fibrosis was induced in C57BL/6 mice by intraperitoneal injection of CCl₄ (2.0 mL/kg of 10% CCl₄ v/v solution in peanut oil) two times per week for eight weeks. Ghrelin (10 μg/kg) was intraperitoneally injected two times per week for eight weeks. A second murine liver fibrosis model was induced by bile duct ligation (BDL) and concurrent ghrelin administration for four weeks. Hematoxylin eosin (H&E), and Masson’s trichrome were used to detect pathological changes to liver tissue. Western blotting was used to detect protein levels of transforming growth factor (TGF)-β1, phosphorylated Smad3 (p-Smad3), I-collage, α-smooth muscle actin (α-SMA), matrix metalloproteinases (MMPs) 2, tissue inhibitor of matrix metalloproteinases (TIMPs) 1, phosphorylated NF-κB (p-NF-κB), and microtubule-associated protein light chain 3 (LC3). In addition, qRT-PCR was used to detect mRNA levels of TGF-β1, I-collage, α-SMA, MMP2, TIMP1 and LC3, while levels of TGF-β1, p-Smad3, I-collage, α-SMA, and LC3 were detected immunohistochemically. Levels of aspartate aminotransferase and alanine aminotransferase were significantly decreased by ghrelin treatment. Ghrelin administration also significantly reduced the extent of pathological changes in both murine liver fibrosis models. Expression levels of I-collage and α-SMA in both models were clearly reduced by ghrelin administration. Furthermore, ghrelin treatment decreased protein expression of TGF-β1 and p-Smad3. The protein levels of NF-κB and LC3 were increased in the CCl₄- and BDL-treatment groups but were significantly reduced following ghrelin treatment. In addition, ghrelin inhibited extracellular matrix formation by decreasing NF-κB expression and maintaining the balance between MMP2 and TIMP1. Our results demonstrated that ghrelin attenuates liver fibrosis via inhibition of the TGF-β1/Smad3 and NF-κB signaling pathways, as well as autophagy suppression.     

SB203580—A Potent p38 MAPK Inhibitor Reduces the Profibrotic Bronchial Fibroblasts Transition Associated with Asthma

Subepithelial fibrosis is a component of the remodeling observed in the bronchial wall of patients diagnosed with asthma. In this process, human bronchial fibroblasts (HBFs) drive the fibroblast-to-myofibroblast transition (FMT) in response to transforming growth factor-β₁ (TGF-β₁), which activates the canonical Smad-dependent signaling. However, the pleiotropic properties of TGF-β₁ also promote the activation of non-canonical signaling pathways which can affect the FMT. In this study we investigated the effect of p38 mitogen-activated protein kinase (MAPK) inhibition by SB203580 on the FMT potential of HBFs derived from asthmatic patients using immunocytofluorescence, real-time PCR and Western blotting methods. Our results demonstrate for the first time the strong effect of p38 MAPK inhibition on the TGF-β₁-induced FMT potential throughout the strong attenuation of myofibroblast-related markers: α-smooth muscle actin (α-SMA), collagen I, fibronectin and connexin 43 in HBFs. We suggest the pleiotropic mechanism of SB203580 on FMT impairment in HBF populations by the diminishing of TGF-β/Smad signaling activation and disturbances in the actin cytoskeleton architecture along with the maturation of focal adhesion sites. These observations justify future research on the role of p38 kinase in FMT efficiency and bronchial wall remodeling in asthma.     

Atractylodin Suppresses TGF-β-Mediated Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells and Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic change in alveolar epithelial cells and leads to the irreversible deterioration of pulmonary function. Transforming growth factor-beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in type 2 lung epithelial cells contributes to excessive collagen deposition and plays an important role in IPF. Atractylodin (ATL) is a kind of herbal medicine that has been proven to protect intestinal inflammation and attenuate acute lung injury. Our study aimed to determine whether EMT played a crucial role in the pathogenesis of pulmonary fibrosis and whether EMT can be utilized as a therapeutic target by ATL treatment to mitigate IPF. To address this topic, we took two steps to investigate: 1. Utilization of anin vitro EMT model by treating alveolar epithelial cells (A549 cells) with TGF-β1 followed by ATL treatment for elucidating the underlying pathways, including Smad2/3 hyperphosphorylation, mitogen-activated protein kinase (MAPK) pathway overexpression, Snail and Slug upregulation, and loss of E-cadherin. Utilization of an in vivo lung injury model by treating bleomycin on mice followed by ATL treatment to demonstrate the therapeutic effectiveness, such as, less collagen deposition and lower E-cadherin expression. In conclusion, ATL attenuates TGF-β1-induced EMT in A549 cells and bleomycin-induced pulmonary fibrosis in mice.     

β-Elemene Attenuates Renal Fibrosis in the Unilateral Ureteral Obstruction Model by Inhibition of STAT3 and Smad3 Signaling via Suppressing MyD88 Expression

Renal fibrosis is a chronic pathological process that seriously endangers human health. However, the current therapeutic options for this disease are extremely limited. Previous studies have shown that signaling factors such as JAK2/STAT3, Smad3, and Myd88 play a regulatory role in renal fibrosis, and β-elemene is a plant-derived sesquiterpenoid organic compound that has been shown to have anti-inflammatory, anti-cancer, and immunomodulatory effects. In the present study, the anti-fibrotic effect of β-elemene was demonstrated by in vivo and in vitro experiments. It was shown that β-elemene inhibited the synthesis of extracellular matrix-related proteins in unilateral ureteral obstruction mice, and TGF-β stimulated rat interstitial fibroblast cells, including α-smooth muscle actin, vimentin, and connective tissue growth factor, etc. Further experiments showed that β-elemene reduced the expression levels of the above-mentioned fibrosis-related proteins by blocking the phosphorylation of JAK2/STAT3, Smad3, and the expression or up-regulation of MyD88. Notably, knockdown of MyD88 attenuated the phosphorylation levels of STAT3 and Smad3 in TGF-β stimulated NRK49F cell, which may be a novel molecular mechanism by which β-elemene affects renal interstitial fibrosis. In conclusion, this study elucidated the anti-interstitial fibrosis effect of β-elemene, which provides a new direction for future research and development of drugs related to chronic kidney disease.     

Hepatocarcinogenesis Prevention by Pirfenidone Is PPARγ Mediated and Involves Modification of Nuclear NF-kB p65/p50 Ratio

Targeted therapies for regulating processes such as inflammation, apoptosis, and fibrogenesis might modulate human HCC development. Pirfenidone (PFD) has shown anti-fibrotic and anti-inflammatory functions in both clinical and experimental studies. The aim of this study was to evaluate PPARγ expression and localization in samples of primary human tumors and assess PFD-effect in early phases of hepatocarcinogenic process. Human HCC tissue samples were obtained by surgical resection. Experimental hepatocarcinogenesis was induced in male Fischer-344 rats. TGF-β1 and α-SMA expression was evaluated as fibrosis markers. NF-kB cascade, TNFα, IL-6, and COX-2 expression and localization were evaluated as inflammation indicators. Caspase-3, p53, and PARP-1 were used as apoptosis markers, PCNA for proliferation. Finally, PPARα and PPARγ expression were evaluated to understand the effect of PFD on the activation of such pathways. PPARγ expression was predominantly localized in cytoplasm in human HCC tissue. PFD was effective to prevent histopathological damage and TGF-β1 and α-SMA overexpression in the experimental model. Anti-inflammatory effects of PFD correlate with diminished IKK and decrease in both IkB-phosphorylation/NF-kB p65 expression and p65-translocation into the nucleus. Pro-apoptotic PFD-induced effects are related with p53 expression, Caspase-3 p17 activation, and PARP-1-cleavage. In conclusion, PFD acts as a tumor suppressor by preventing fibrosis, reducing inflammation, and promoting apoptosis in MRHM.     

Nuclear Receptor Nur77 Controls Cardiac Fibrosis through Distinct Actions on Fibroblasts and Cardiomyocytes

Fibrosis is a hallmark of adverse cardiac remodeling, which promotes heart failure, but it is also an essential repair mechanism to prevent cardiac rupture, signifying the importance of appropriate regulation of this process. In the remodeling heart, cardiac fibroblasts (CFs) differentiate into myofibroblasts (MyoFB), which are the key mediators of the fibrotic response. Additionally, cardiomyocytes are involved by providing pro-fibrotic cues. Nuclear receptor Nur77 is known to reduce cardiac hypertrophy and associated fibrosis; however, the exact function of Nur77 in the fibrotic response is yet unknown. Here, we show that Nur77-deficient mice exhibit severe myocardial wall thinning, rupture and reduced collagen fiber density after myocardial infarction and chronic isoproterenol (ISO) infusion. Upon Nur77 knockdown in cultured rat CFs, expression of MyoFB markers and extracellular matrix proteins is reduced after stimulation with ISO or transforming growth factor–β (TGF-β). Accordingly, Nur77-depleted CFs produce less collagen and exhibit diminished proliferation and wound closure capacity. Interestingly, Nur77 knockdown in neonatal rat cardiomyocytes results in increased paracrine induction of MyoFB differentiation, which was blocked by TGF-β receptor antagonism. Taken together, Nur77-mediated regulation involves CF-intrinsic promotion of CF-to-MyoFB transition and inhibition of cardiomyocyte-driven paracrine TGF-β-mediated MyoFB differentiation. As such, Nur77 provides distinct, cell-specific regulation of cardiac fibrosis.     

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.     

Oxidative Stress Enhances the TGF-β2-RhoA-MRTF-A/B Axis in Cells Entering Endothelial-Mesenchymal Transition

Around 45% of deaths in the EU and the US are due to fibrotic diseases. Although myofibroblasts are detected in various fibrotic tissues, they are mostly transdifferentiated from endothelial cells during the endothelial-mesenchymal transition (EndMT) induced by tumor growth factor-beta (TGF-β) family members. Growing evidence indicates that oxidative stress might enhance the sensitivity and the effects of TGF-β stimulation; however, the molecular mechanisms involved in the coordination of oxidative stress and TGF-β inductions remain poorly understood. Our findings indicate for the first time that oxidative stress enhances mesenchymal trans-differentiation of human microvascular endothelial cells (HMEC-1 cells) and that the oxidative stress-dependent TGF-β2-RhoA/Rac1-MRTF-A axis is critical for the induction of later stages of EndMT. This additive effect was manifested in TGF-β1-stimulated and Snail-overexpressed cells, where it caused higher cell elongation and faster migration on collagen I layers. Additionally, Western blot assay indicated the presence of alterations in cell contraction and EndMT markers. We conclude that complex anti-fibrotic therapies based on the inhibition of MRTF activities and oxidative stress might be an attractive target for fibrosis treatment.     

Chrysin Ameliorates Cyclosporine-A-Induced Renal Fibrosis by Inhibiting TGF-β1-Induced Epithelial–Mesenchymal Transition

Cyclosporine A (CsA) is a nephrotoxicant that causes fibrosis via induction of epithelial–mesenchymal transition (EMT). The flavonoid chrysin has been reported to have anti-fibrotic activity and inhibit signaling pathways that are activated during EMT. This study investigated the nephroprotective role of chrysin in the prevention of CsA-induced renal fibrosis and elucidated a mechanism of inhibition against CsA-induced EMT in proximal tubule cells. Treatment with chrysin prevented CsA-induced renal dysfunction in Sprague Dawley rats measured by blood urea nitrogen (BUN), serum creatinine and creatinine clearance. Chrysin inhibited CsA-induced tubulointerstitial fibrosis, characterized by reduced tubular damage and collagen deposition. In vitro, chrysin significantly inhibited EMT in LLC-PK₁ cells, evidenced by inhibition of cell migration, decreased collagen expression, reduced presence of mesenchymal markers and elevated epithelial junction proteins. Furthermore, chrysin co-treatment diminished CsA-induced TGF-β₁ signaling pathways, decreasing Smad 3 phosphorylation which lead to a subsequent reduction in Snail expression. Chrysin also inhibited activation of the Akt/ GSK-3β pathway. Inhibition of both pathways diminished the cytosolic accumulation of β-catenin, a known trigger for EMT. In conclusion, flavonoids such as chrysin offer protection against CsA-induced renal dysfunction and interstitial fibrosis. Chrysin was shown to inhibit CsA-induced TGF-β₁-dependent EMT in proximal tubule cells by modulation of Smad-dependent and independent signaling pathways.     

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1-Evoked Renal Tubular Epithelial Cells

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention.     

Corilagin Attenuates Aerosol Bleomycin-Induced Experimental Lung Injury

Idiopathic pulmonary fibrosis (IPF) is a progressing lethal disease with few clinically effective therapies. Corilagin is a tannin derivative which shows anti-inflammatory and antifibrotics properties and is potentiated in treating IPF. Here, we investigated the effect of corilagin on lung injury following bleomycin exposure in an animal model of pulmonary fibrosis. Corilagin abrogated bleomycin-induced lung fibrosis as assessed by H&E; Masson’s trichrome staining and lung hydroxyproline content in lung tissue. Corilagin reduced the number of apoptotic lung cells and prevented lung epithelial cells from membrane breakdown, effluence of lamellar bodies and thickening of the respiratory membrane. Bleomycin exposure induced expression of MDA, IKKα, phosphorylated IKKα (p-IKKα), NF-κB P65, TNF-α and IL-1β, and reduced I-κB expression in mice lung tissue or in BALF. These changes were reversed by high-dose corilagin (100 mg/kg i.p) more dramatically than by low dose (10 mg/kg i.p). Last, corilagin inhibits TGF-β1 production and α-SMA expression in lung tissue samples. Taken together, these findings confirmed that corilagin attenuates bleomycin-induced epithelial injury and fibrosis via inactivation of oxidative stress, proinflammatory cytokine release and NF-κB and TGF-β1 signaling. Corilagin may serve as a promising therapeutic agent for pulmonary fibrosis.     

The Fibrotic Effects of TMAO on Human Renal Fibroblasts Is Mediated by NLRP3, Caspase-1 and the PERK/Akt/mTOR Pathway

Trimethylamine N-oxide (TMAO), a product of gut microbiota metabolism, has previously been shown to be implicated in chronic kidney disease. A high TMAO-containing diet has been found to cause tubulointerstitial renal fibrosis in mice. However, today there are no data linking specific molecular pathways with the effect of TMAO on human renal fibrosis. The aim of this study was to investigate the fibrotic effects of TMAO on renal fibroblasts and to elucidate the molecular pathways involved. We found that TMAO promoted renal fibroblast activation and fibroblast proliferation via the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 signaling. We also found that TMAO increased the total collagen production from renal fibroblasts via the PERK/Akt/mTOR pathway. However, TMAO did not induce fibronectin or TGF-β1 release from renal fibroblasts. We have unraveled that the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 mediates TMAO’s fibrotic effect on human renal fibroblasts. Our results can pave the way for future research to further clarify the molecular mechanism behind TMAO’s effects and to identify novel therapeutic targets in the context of chronic kidney disease.     

Renoprotective Effects of Alpha-1 Antitrypsin against Tacrolimus-Induced Renal Injury

The protective effects of alpha-1 antitrypsin (AAT) in tacrolimus (TAC)-induced renal injury was evaluated in a rat model. The TAC group rats were subcutaneously injected with 2 mg/kg TAC every day for four weeks. The TAC with AAT group was cotreated with daily subcutaneous injections of TAC and intraperitoneal injections of AAT (80 mg/kg) for four weeks. The effects of AAT on TAC-induced renal injury were evaluated using serum biochemistry, histopathology, and Western blotting. The TAC injection significantly increased renal interstitial fibrosis, inflammation, and apoptosis as compared to the control treatment. The histopathological examination showed that cotreatment of TAC and AAT attenuated interstitial fibrosis (collagen, fibronectin, and α-SMA staining), and α-SMA expression in Western blotting was also decreased. Immunohistochemical staining for inflammation (osteopontin and ED-1 staining) revealed improved interstitial inflammation in the TAC with AAT group compared to that in the TAC group. The TAC treatment increased renal apoptosis compared to the control treatment, based on the results of increased immunohistochemical staining of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), increased caspase-3 activity, and lower Bcl-2 to Bad expression ratio. However, AAT cotreatment significantly changed these markers and consequently showed decreased apoptosis. AAT protects against TAC-induced renal injury via antifibrotic, anti-inflammatory, and antiapoptotic effects.     

WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-β and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-β-activated human cardiac fibroblasts. We found that WNT3a and TGF-β induced a β-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-β triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the β-catenin pathway with the GSK3β inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-β, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-β-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-β and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-β-induced production of collagen I. In conclusion, WNT/β-catenin signaling promotes TGF-β-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.