Targeted inhibition of transforming growth factor-ß type I receptor by AZ12601011 improves paraquat poisoning-induced multiple organ fibrosis.

Paraquat (PQ) causes fatal poisoning that leads to systemic multiple organ fibrosis, and transforming growth factor (TGF)-ß1 plays a critical role in this process. In this study, we aimed to investigate the effects of AZ12601011 (a small molecular inhibitor of TGFßRI) on PQ-induced multiple organ fibrosis. We established a mouse model of PQ in vivo and used PQ-treated lung epithelial cell (A549) and renal tubular epithelial cells (TECs) in vitro. Haematoxylin-eosin and Masson staining revealed that AZ12601011 ameliorated pulmonary, hepatic, and renal fibrosis, consistent with the decrease in the levels of fibrotic indicators, alpha-smooth muscle actin (α-SMA) and collagen-1, in the lungs and kidneys of PQ-treated mice. In vitro data showed that AZ12601011 suppressed the induction of α-SMA and collagen-1 in PQ-treated A549 cells and TECs. In addition, AZ12601011 inhibited the release of inflammatory factors, interleukin (IL)-1ß, IL-6, and tumour necrosis factor-α. Mechanistically, TGF-ß and TGFßRI levels were significantly upregulated in the lungs and kidneys of PQ-treated mice. Cellular thermal shift assay and western blotting revealed that AZ12601011 directly bound with TGFßRI and blocked the activation of Smad3 downstream. In conclusion, our findings revealed that AZ12601011 attenuated PQ-induced multiple organ fibrosis by blocking the TGF-ß/Smad3 signalling pathway, suggesting its potential for PQ poisoning treatment.

LncRNA GAS5 protects against TGF-ß-induced renal fibrosis via the Smad3/miRNA-142-5p axis.

Increasing evidence shows that long noncoding RNAs (lncRNAs) play an important role in kidney disease. In this study, we investigated the role of the lncRNA growth arrest-specific 5 (GAS5) in the pathogenesis of renal fibrosis. We found that GAS5 was markedly decreased in the fibrotic kidney of a unilateral ureteral obstructive nephropathy mouse model. In addition, GAS5 was expressed in mouse tubular epithelial cells (mTECs) and interstitial fibroblasts in normal renal tissue and was especially highly expressed in the cytoplasm. In vitro experiments showed that GAS5 was downregulated by transforming growth factor-ß1 (TGF-ß1) in a dose- and time-dependent manner. Overexpression of GAS5 blocked TGF-ß1-induced collagen type I and fibronectin expression and vice versa. Mechanistic experiments revealed that Smad3 but not Smad2 drove the regulation of GAS5. More importantly, GAS5 interacted with miR-142-5p and was involved in the renoprotective effect by participating in the competing endogenous RNA network. Finally, we also found that knockdown of GAS5 promoted TGF-ß1-induced mouse tubular epithelial cell apoptosis via the Smad3 pathway. Taken together, our results uncovered a lncRNA/miRNA competing endogenous RNA network-based mechanism that modulates extracellular matrix formation and cell apoptosis via the Smad3 pathway.NEW & NOTEWORTHY In this work, we mainly discuss long noncoding RNA growth arrest-specific 5 (GAS5), acting in a renoprotective role via the Smad3/miRNA-142-5p axis, that modulates extracellular matrix formation and cell apoptosis. Overexpression of GAS5 effectively blocked renal fibrosis in vitro. This study reveals that GAS5 may represent as a novel and precision therapeutic target for alleviating renal fibrosis.

CircHIPK3 regulates pulmonary fibrosis by facilitating glycolysis in miR-30a-3p/FOXK2-dependent manner.

Pulmonary fibrosis develops when myofibroblasts and extracellular matrix excessively accumulate in the injured lung, but what drives fibrosis is not fully understood. Glycolysis has been linked to cell growth and proliferation, and several studies have shown enhanced glycolysis promotes pulmonary fibrosis. However, detailed studies describing this switch remain limited. Here, we identified that TGF-ß1 effectively increased the expression of circHIPK3 in lung fibroblasts, and circHIPK3 inhibition attenuated the activation, proliferation, and glycolysis of fibroblasts in vitro. Dual-luciferase reporter gene assays, RNA immunoprecipitation (RIP), and RNA pull-down assays showed that circHIPK3 could function as a sponge of miR-30a-3p and inhibit its expression. Furthermore, FOXK2, a driver transcription factor of glycolysis, was identified to be a direct target of miR-30a-3p. Mechanistically, circHIPK3 could enhance the expression of FOXK2 via sponging miR-30a-3p, thereby facilitating fibroblast glycolysis and activation. Besides, miR-30a-3p overexpression or FOXK2 knockdown blocked fibroblast activation induced by TGF-ß1 and abrogated the profibrotic effects of circHIPK3. Moreover, circHIPK3 and miR-30a-3p were also dysregulated in fibrotic murine lung tissues induced by silica. Adeno-associated virus (AAV)-mediated circHIPK3 silence or miR-30a-3p overexpression alleviated silica-induced pulmonary fibrosis in vivo. In conclusion, our results identified circHIPK3/miR-30a-3p/FOXK2 regulatory pathway as an important glycolysis cascade in pulmonary fibrosis.

RS4651 suppresses lung fibroblast activation via the TGF-ß1/SMAD signalling pathway.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive disease resulting in respiratory failure with no efficient treatment options. We investigated the protective effect of RS4651 on pulmonary fibrosis in mice and the mechanism. METHODS: Intratracheal injection of bleomycin (BLM) was used to induce pulmonary fibrosis in mice. RS4561 was administered intraperitoneally at different doses. Histopathological changes were observed. The level of alpha-smooth muscle actin (α-SMA) were also tested. In vitro, the proliferation and migratory effects of RS4651 treatment on MRC-5 cells pre-treated with transforming growth factor (TGF-ß1) were examined. RNA-sequencing was used to detect differentially expressed target genes. Then, the expression of α-SMA, pSMAD2 and SMAD7 were analysed during RS4651 treatment of MRC-5 cells with or without silencing by SMAD7 siRNA. RESULTS: Histopathological staining results showed decreased collagen deposition in RS4651 administered mice. Additionally, a lower level of α-SMA was also observed compared to the BLM group. The results of in vitro studies confirmed that RS4651 can inhibit the proliferation and migration, as well as α-SMA and pSMAD2 expression in MRC-5 cells treated with TGF-ß1. RNA-sequencing data identified the target gene SMAD7. We found that RS4651 could upregulate SMAD7 expression and inhibit the proliferation and migration of MRC-5 cells via SMAD7, and RS4651 inhibition of α-SMA and pSMAD2 expression was blocked in SMAD7-siRNA MRC-5 cells. In vivo studies further confirmed that RS4651 could upregulate SMAD7 expression in BLM-induced lung fibrosis in mice. CONCLUSIONS: Our data suggest that RS4651 alleviates BLM-induced pulmonary fibrosis in mice by inhibiting the TGF-ß1/SMAD signalling pathway.

AITC inhibits fibroblast-myofibroblast transition via TRPA1-independent MAPK and NRF2/HO-1 pathways and reverses corticosteroids insensitivity in human lung fibroblasts.

BACKGROUND: Little is known on the role of transient receptor potential ankyrin 1 (TRPA1) in fibroblast-myofibroblast transition (FMT) that can lead to airway remodeling which is a major problem for severe asthma and fibrosis. Thus, this study investigated the effect of TRPA1 modulators on transforming growth factor beta 1(TGF-ß1) -treated lung fibroblasts. METHODS: MRC-5 cells were preincubated with TGF-ß1 for 24 h. TRPA1 agonist or antagonist were added and further incubated for 24 h. The changes in TRPA1 and alpha-smooth muscle actin (α-SMA) expressions by stimuli were evaluated using qRT-PCR, western blot and immunohistochemical analyses. Statistical significance was determined by using one- or two-way ANOVA, followed by Bonferroni's post hoc analysis for comparison of multiple groups and paired 2-tailed Student's t-test between 2 groups. RESULTS: MRC-5 cells treated by TGF-ß1 significantly upregulated α-SMA mRNA expressions (P < 0.01), but downregulated TRPA1 gene expression (P < 0.001). Post-treatment of TRPA1 activator, allyl isothiocyanate (AITC), after TGF-ß1 significantly downregulated the α-SMA gene induction (P < 0.01 at 24 h), protein expression (P < 0.05) and immunoreactivity with stress fibers (P < 0.05). On the other hand, TRPA1 antagonist HC-030031 did not prevent this effect, and instead tended to facilitate the suppressive effect of AITC when co-stimulated. AITC significantly increased phosphorylated- extracellular signal-regulated kinase (ERK) 1/2 and heme oxygenase (HO)-1 protein expressions (P < 0.05) in TGF-ß1-treated cells. Combined inhibition with ERK1/2 mitogen-activated protein kinase (MAPK) and nuclear factor erythroid 2-related factor (NRF2) almost completely reversed AITC-induced α-SMA suppression (P < 0.05). Dexamethasone was not able to inhibit the upregulated α-SMA induction by TGF-ß1. However, AITC improved dexamethasone-insensitive myodifferentiation in the presence of the corticosteroid (P < 0.01). CONCLUSION: We found that AITC exerts protective effect on TGF-ß1-induced α-SMA induction by activating ERK1/2 MAPK and NRF2/HO-1 pathways in lung fibroblasts. It also overcomes corticosteroids insensitivity in TGF-ß1-induced α-SMA induction. TRPA1 antagonist modulates the suppressive effect, but not prevent it. AITC and TRPA1 antagonist may be therapeutic agents in treating chronic respiratory diseases.

Hard antler extract inhibits invasion and epithelial-mesenchymal transition of triple-negative and Her-2+ breast cancer cells by attenuating nuclear factor-κB signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Hard antler extract (HAE) is a traditional Chinese medicine and has potent antitumor, antioxidative, anti-inflammatory, and immunomodulatory activities. Previous studies have demonstrated that HAE can inhibit human prostate cancer metastasis and murine breast cancer proliferation. However, the effect of HAE on human breast cancer cells has not been clarified. AIM OF THE STUDY: To investigate the effects and underlying mechanism of HAE on self-renewal of stem-like cells and spontaneous and transforming growth factor (TGF)-ß1-enhanced wound healing, invasion and epithelial-mesenchymal transition (EMT) in breast cancer cells. METHODS: HAE was prepared from sika deer by sequential enzymatic digestions and the active compounds were determined by HPLC. The effects of HAE on the viability, mammosphere formation, wound healing and invasion of MDA-MB-231 and SK-BR3 cells were determined. The impact of HAE treatment on spontaneous and TGF-ß1-promoted EMT and the nuclear factor (NF)-κB signaling in breast cancer cells was examined by quantitative RT-PCR and western blotting. RESULTS: Treatment with HAE at varying concentrations did not change the viability of breast cancer cells. However, HAE at 0.25 or 0.5 mg/mL significantly reduced the number and size of formed mammospheres, and inhibited spontaneous and TGF-ß1-enhanced wound healing, invasion and EMT in MDA-MB-231 and SK-BR3 cells in a dose-dependent manner. TGF-ß1 treatment significantly decreased IκBα expression and increased NF-kBp65 phosphorylation in breast cancer cells, indicating that TGF-ß1 enhanced NF-κB signaling. In contrast, HAE treatment attenuated the spontaneous and TGF-ß1-enhanced NF-κB signaling in breast cancer cells. CONCLUSION: Our data indicated that HAE inhibited the self-renewal of stem-like cells and spontaneous and TGF-ß1-enhanced wound healing, invasion and EMT in breast cancer cells by attenuating the NF-κB signaling in vitro.

ELAV Proteins Bind and Stabilize C/EBP mRNA in the Induction of Long-Term Memory in Aplysia.

Long-term memory (LTM) formation is a critical survival process by which an animal retains information about prior experiences to guide future behavior. In the experimentally advantageous marine mollusk Aplysia, LTM for sensitization can be induced by the presentation of two aversive shocks to the animal's tail. Each of these training trials recruits distinct growth factor signaling systems that promote LTM formation. Specifically, whereas intact TrkB signaling during Trial 1 promotes an initial and transient increase of the immediate early gene apc/ebp mRNA, a prolonged increase in apc/ebp gene expression required for LTM formation requires the addition of TGFß signaling during Trial 2. Here we explored the molecular mechanisms by which Trial 2 achieves the essential prolonged gene expression of apc/ebp We find that this prolonged gene expression is not dependent on de novo transcription, but that apc/ebp mRNA synthesized by Trial 1 is post-transcriptionally stabilized by interacting with the RNA-binding protein ApELAV. This interaction is promoted by p38 MAPK activation initiated by TGFß. We further demonstrate that blocking the interaction of ApELAV with its target mRNA during Trial 2 blocks both the prolonged increase in apc/ebp gene expression and the behavioral induction of LTM. Collectively, our findings elucidate both when and how ELAV proteins are recruited for the stabilization of mRNA in LTM formation. Stabilization of a transiently expressed immediate early gene mRNA by a repeated training trial may therefore serve as a "filter" for learning, permitting only specific events to cause lasting transcriptional changes and behavioral LTM.SIGNIFICANCE STATEMENT: In the present paper, we significantly extend the general field of molecular processing in long-term memory (LTM) by describing a novel form of pretranslational processing required for LTM, which relies on the stabilization of a newly synthesized mRNA by a class of RNA binding proteins (ELAVs). There are now compelling data showing that important processing can occur after transcription of a gene, but before translation of the message into protein. Although the potential importance of ELAV proteins in LTM formation has previously been reported, the specific actions of ELAV proteins during LTM formation remained to be understood. Our new findings thus complement and extend this literature by demonstrating when and how this post-transcriptional gene regulation is mediated in the induction of LTM.

Dexamethasone rescues TGF-ß1-mediated ß2-adrenergic receptor dysfunction and attenuates phosphodiesterase 4D expression in human airway smooth muscle cells.

Glucocorticoids (GCs) and ß2-adrenergic receptor (ß2AR) agonists improve asthma outcomes in most patients. GCs also modulate gene expression in human airway smooth muscle (HASM), thereby attenuating airway inflammation and airway hyperresponsiveness that define asthma. Our previous studies showed that the pro-fibrotic cytokine, transforming growth factor- ß1 (TGF-ß1) increases phosphodiesterase 4D (PDE4D) expression that attenuates agonist-induced levels of intracellular cAMP. Decreased cAMP levels then diminishes ß2 agonist-induced airway relaxation. In the current study, we investigated whether glucocorticoids reverse TGF-ß1-effects on ß2-agonist-induced bronchodilation and modulate pde4d gene expression in HASM. Dexamethasone (DEX) reversed TGF-ß1 effects on cAMP levels induced by isoproterenol (ISO). TGF-ß1 also attenuated G protein-dependent responses to cholera toxin (CTX), a Gαs stimulator downstream from the ß2AR receptor. Previously, we demonstrated that TGF-ß1 treatment increased ß2AR phosphorylation to induce hyporesponsiveness to a ß2 agonist. Our current data shows that expression of grk2/3, kinases associated with attenuation of ß2AR function, are not altered with TGF-ß1 stimulation. Interestingly, DEX also attenuated TGF-ß1-induced pde4d gene expression. These data suggest that steroids may be an effective therapy for treatment of asthma patients whose disease is primarily driven by elevated TGF-ß1 levels.

Effect of selective inhibition or activation of PGE2 EP1 receptor on glomerulosclerosis.

Prostaglandin E2 (PGE2) is involved in numerous physiological and pathological processes of the kidney via its four receptors. A previous study has suggested that a defect in the PGE2 receptor 1 (EP1) gene markedly suppressed the transforming growth factor­ß1 (TGF­ß1)­induced mesangial cell (MC) proliferation and extracellular matrix aggregation. Therefore, the present study aimed to adopt a pharmacological method of specifically suppressing or activating the EP1 receptor to further verify and demonstrate these results. The EP1 receptor antagonist SC­19220 and EP1 receptor agonist 17­phenyl­trinor­PGE2 ethyl amide (17­pt­PGE2) were selectively used to treat five­sixths nephrectomy renal fibrosis model mice and TGF­ß1­stimulated MCs. An Alpha screen PGE2 assay kit, flow cytometry, western blotting and immunohistochemical techniques were adopted to perform in vivo and in vitro experiments. The present results suggested that compared with the control group, the selective EP1 receptor antagonist SC­19220 improved renal function, markedly reduced the plasma blood urea nitrogen and creatinine levels (P<0.05) and alleviated glomerulosclerosis (P<0.05). By contrast, the EP1 receptor agonist 17­pt­PGE2 aggravated renal dysfunction and glomerulosclerosis (P<0.05). To verify the renal protection mechanisms mediated by suppression of the EP1 receptor, the expression levels of endoplasmic reticulum stress (ERS)­related proteins, including chaperone glucose­regulated protein 78 (GRP78), transient receptor potential channel 1 (TRPC1) and protein kinase R­like endoplasmic reticulum kinase (PERK), were further evaluated histologically. The expression of GRP78, TRPC1 and PERK in the antagonist treatment group were markedly downregulated (P<0.05), whereas those in the agonist treatment group were upregulated (P<0.05). The present in vitro experiments demonstrated that, compared with the control group, the EP1 receptor antagonist suppressed the expression of GRP78, TRPC1 and PERK (P<0.05), reduced the production of PGE2 (P<0.05) and decreased the MC apoptosis rate (P<0.05), thus alleviating TGF­ß1­stimulated MC injury. Consequently, consistent with previous results, selectively antagonizing the EP1 receptor improved renal function and mitigated glomerulosclerosis, and its potential mechanism might be associated with the suppression of ERS.

Upregulation of miRNA-1228-3p alleviates TGF-ß-induced fibrosis in renal tubular epithelial cells.

BACKGROUND: Chronic kidney disease (CKD) has become a major public health issue, which can lead to renal fibrosis regardless of the initial injury. It has been previously reported that miRNA-1228-3p was correlate with the progression of kidney fibrosis. However, the mechanism by which miRNA-1228-3p regulates renal fibrosis remains unclear. METHODS: Renal tubular epithelial cells (HK-2) were treated with TGF-ß1 (10 ng/ml) in an in vitro model of renal fibrosis. Gene and protein expressions in HK-2 cells were measured by Western-blot and RT-qPCR, respectively. The relation between miRNA-1228-3p and its target gene was investigated by dual luciferase report analysis. RESULTS: Upregulation of miRNA-1228-3p significantly inhibited TGF-ß1-induced fibrosis of HK-2 cells in vitro by targeting GDF11. In addition, miRNA-1228-3p exhibited anti-fibrosis effect through inhibition of the smad2/smad4 signaling pathway. CONCLUSION: Upregulation of miRNA-1228-3p markedly inhibited the progression of renal fibrosis in vitro, indicating that miRNA-1228-3p may serve as a potential novel target for the treatment of renal fibrosis.

Prolonged Scar-in-a-Jar: an in vitro screening tool for anti-fibrotic therapies using biomarkers of extracellular matrix synthesis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapidly progressing disease with challenging management. To find novel effective therapies, better preclinical models are needed for the screening of anti-fibrotic compounds. Activated fibroblasts drive fibrogenesis and are the main cells responsible for the accumulation of extracellular matrix (ECM). Here, a prolonged Scar-in-a-Jar assay was combined with clinically validated biochemical markers of ECM synthesis to evaluate ECM synthesis over time. To validate the model as a drug screening tool for novel anti-fibrotic compounds, two approved compounds for IPF, nintedanib and pirfenidone, and a compound in development, omipalisib, were tested. METHODS: Primary human lung fibroblasts from healthy donors were cultured for 12 days in the presence of ficoll and were stimulated with TGF-ß1 with or without treatment with an ALK5/TGF-ß1 receptor kinase inhibitor (ALK5i), nintedanib, pirfenidone or the mTOR/PI3K inhibitor omipalisib (GSK2126458). Biomarkers of ECM synthesis were evaluated over time in cell supernatants using ELISAs to assess type I, III, IV, V and VI collagen formation (PRO-C1, PRO-C3, PRO-C4, PRO-C5, PRO-C6), fibronectin (FBN-C) deposition and α-smooth muscle actin (α-SMA) expression. RESULTS: TGF-ß1 induced synthesis of PRO-C1, PRO-C6 and FBN-C as compared with unstimulated fibroblasts at all timepoints, while PRO-C3 and α-SMA levels were not elevated until day 8. Elevated biomarkers were reduced by suppressing TGF-ß1 signalling with ALK5i. Nintedanib and omipalisib were able to reduce all biomarkers induced by TGF-ß1 in a concentration dependent manner, while pirfenidone had no effect on α-SMA. CONCLUSIONS: TGF-ß1 stimulated synthesis of type I, III and VI collagen, fibronectin and α-SMA but not type IV or V collagen. Synthesis was increased over time, although temporal profiles differed, and was modulated pharmacologically by ALK5i, nintedanib, pirfenidone and omipalisib. This prolonged 12-day Scar-in-a-Jar assay utilising biochemical markers of ECM synthesis provides a useful screening tool for novel anti-fibrotic compounds.

WNT/ß-catenin signal inhibitor IC-2-derived small-molecule compounds suppress TGF-ß1-induced fibrogenic response of renal epithelial cells by inhibiting SMAD2/3 signalling.

Renal fibrosis compromises kidney function, and it is a risk factor for chronic kidney disease (CKD). CKD ultimately progresses to end-stage kidney disease that can be cured only by kidney transplantation. Owing to the increasing number of CKD patients, effective treatment strategies are urgently required for renal fibrosis. TGF-ß is a well-established fibrogenic factor that signals through SMAD2/3 signaling pathway. It was shown that there is a cross-talk between TGF-ß/SMAD and WNT/ß-catenin signaling pathways in renal tubular epithelial cells, and that a WNT/ß-catenin inhibitor, ICG-001, ameliorates TGF-ß1induced renal fibrosis. IC-2, a derivative of ICG-001, has been shown to potently induce hepatocyte differentiation of human mesenchymal stem cells by inhibiting WNT/ß-catenin signaling. In the present study, we examined the effect of ICG-001, IC-2, and IC-2 derivatives (IC-2-506-1, IC-2-506-2, IC-2-506-3, IC-2-Ar-Cl, IC-2-OH, IC-2-OTBS, and IC-2-F) on TGF-ß1-induced SMAD activation and fibrogenic response in immortalized human renal tubular epithelial HK-2 cells. All these compounds inhibited LiCl-induced WNT/ß-catenin reporter activation to a similar extent, whereas ICG-001, IC-2-OTBS, and IC-2-F almost completely suppressed TGF-ß1-induced SMAD reporter activation without apparent cytotoxicity. Phosphorylation of SMAD2/3 by TGF-ß1 was more potently inhibited by IC-2-OTBS and IC-2-F than by ICG-001 and IC-2. IC-2-F suppressed TGF-ß1-induced COL1A1 protein expression, whereas IC-2-506-1 and IC-2-OTBS suppressed TGF-ß1-induced epithelial-mesenchymal transition. These results demonstrated that IC-2 derivatives suppress the TGF-ß1-induced fibrogenic response of tubular epithelial cells and thus could be promising therapeutic agents for the treatment of renal fibrosis.

Telmisartan inhibits oxalate and calcium oxalate crystal-induced epithelial-mesenchymal transformation via PPAR-γ-AKT/STAT3/p38 MAPK-Snail pathway.

AIMS: Telmisartan (TLM), a highly selective angiotensin II type 1 receptor blocker (ARB) and partial PPAR-γ agonist, has versatile beneficial effects against oxidative stress, apoptosis, inflammatory responses and epithelial-mesenchymal transition (EMT). However, its underlying mechanism of inhibiting oxalate and calcium oxalate (CaOx) crystal-induced EMT by activating the PPAR-γ pathway remains unclear. MAIN METHODS: CCK-8 assays were used to evaluate the effects of TLM on cell viability. In addition, intracellular reactive oxygen species (ROS) levels were measured by the cell-permeable fluorogenic probe 2,7-dichlorofluorescein diacetate (DCFH-DA). Wound-healing and Transwell assays were used to evaluate the migration ability of HK2 cells exposed to oxalate. Moreover, immunofluorescence, immunohistochemistry and western blotting were used to examine the expression of E-cadherin, N-cadherin, vimentin and α-SMA and explore the underlying molecular mechanisms in HK2 cells and a stone-forming rat model. KEY FINDINGS: Our results showed that TLM treatment could protect HK2 cells from oxalate-induced cytotoxicity and oxidative stress injury. Additionally, TLM prevented EMT induction by oxalate and CaOx crystals via the PPAR-γ-AKT/STAT3/p38 MAPK-Snail pathway in vitro and in vivo. However, knockdown of PPAR-γ with small interfering RNA or the PPAR-γ-specific antagonist GW9662 abrogated these protective effects of TLM. SIGNIFICANCE: As a PPAR-γ agonist, TLM can ameliorate oxalate and CaOx crystal-induced EMT by exerting an antioxidant effect through the PPAR-γ-AKT/STAT3/p38 MAPK-Snail signaling pathway. Therefore, TLM can block EMT progression and could be a potential therapeutic agent for preventing and treating calcium oxalate urolithiasis formation and recurrence.

YY1 mediates TGF-ß1-induced EMT and pro-fibrogenesis in alveolar epithelial cells.

Pulmonary fibrosis is a chronic, progressive lung disease associated with lung damage and scarring. The pathological mechanism causing pulmonary fibrosis remains unknown. Emerging evidence suggests prominent roles of epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AECs) in myofibroblast formation and progressive pulmonary fibrosis. Our previous work has demonstrated the regulation of YY1 in idiopathic pulmonary fibrosis and pathogenesis of fibroid lung. However, the specific function of YY1 in AECs during the pathogenesis of pulmonary fibrosis is yet to be determined. Herein, we found the higher level of YY1 in primary fibroblasts than that in primary epithelial cells from the lung of mouse. A549 and BEAS-2B cells, serving as models for type II alveolar pulmonary epithelium in vitro, were used to determine the function of YY1 during EMT of AECs. TGF-ß-induced activation of the pro-fibrotic program was applied to determine the role YY1 may play in pro-fibrogenesis of type II alveolar epithelial cells. Upregulation of YY1 was associated with EMT and pro-fibrotic phenotype induced by TGF-ß treatment. Targeted knockdown of YY1 abrogated the EMT induction by TGF-ß treatment. Enforced expression of YY1 can partly mimic the TGF-ß-induced pro-fibrotic change in either A549 cell line or primary alveolar epithelial cells, indicating the induction of YY1 expression may mediate the TGF-ß-induced EMT and pro-fibrosis. In addition, the translocation of NF-κB p65 from the cytoplasm to the nucleus was demonstrated in A549 cells after TGF-ß treatment and/or YY1 overexpression, suggesting that NF-κB-YY1 signaling pathway regulates pulmonary fibrotic progression in lung epithelial cells. These findings will shed light on the better understanding of mechanisms regulating pro-fibrogenesis in AECs and pathogenesis of lung fibrosis.

rAAV6-mediated miR-29b delivery suppresses renal fibrosis.

BACKGROUND: Previous studies showed that microRNA-29b (miR-29b) inhibits renal fibrosis. Therefore, miR-29b replacement therapy represents a promising approach for treating renal fibrosis. However, an efficient method of kidney-targeted miRNA delivery has yet to be established. Recombinant adeno-associated virus (rAAV) vectors have great potential for clinical application. For kidney-targeted gene delivery, the most suitable AAV serotype has yet to be established. Here, we identified the most suitable AAV serotype for kidney-targeted gene delivery and determined that AAV-mediated miR-29b delivery can suppress renal fibrosis in vivo. METHOD: To determine which AAV serotype is suitable for kidney cells, GFP-positive cells were identified by flow cytometry after the infection of rAAV serotype 1-9 vectors containing the EGFP gene. Next, we injected rAAV vectors into the renal pelvis to determine transduction efficiency in vivo. GFP expression was measured seven days after injecting rAAV serotype 1-9 vectors carrying the EGFP gene. Finally, we investigated whether rAAV6-mediated miR-29b delivery can suppress renal fibrosis in UUO mouse model. RESULTS: We found that rAAV6 vector is the most suitable for targeting kidney cells regardless of animal species in vitro and rAAV6 is the most suitable vector for kidney-targeted in vivo gene delivery in mice. Intra-renal pelvic injection of rAAV vectors can transduce genes into kidney TECs. Furthermore, rAAV6-mediated miR-29b delivery attenuated renal fibrosis in UUO model by suppressing Snail1 expression. CONCLUSION: Our study has revealed that rAAV6 is the most suitable serotype for kidney-targeted gene delivery and rAAV6-mediated miR-29b delivery into kidney TECs can suppress established renal fibrosis.

Pirfenidone attenuates lung fibrotic fibroblast responses to transforming growth factor-ß1.

BACKGROUND: Pirfenidone, an antifibrotic agent used for the treatment of idiopathic pulmonary fibrosis (IPF), functions by inhibiting myofibroblast differentiation, which is involved in transforming growth factor (TGF)-ß1-induced IPF pathogenesis. However, unlike normal lung fibroblasts, the relationship between pirfenidone responses of TGF-ß1-induced human fibrotic lung fibroblasts and lung fibrosis has not been elucidated. METHODS: The effects of pirfenidone were evaluated in lung fibroblasts isolated from fibrotic human lung tissues after TGF-ß1 exposure. The ability of two new pharmacological targets of pirfenidone, collagen triple helix repeat containing protein 1(CTHRC1) and four-and-a-half LIM domain protein 2 (FHL2), to mediate contraction of collagen gels and migration toward fibronectin were assessed in vitro. RESULTS: Compared to control lung fibroblasts, pirfenidone significantly restored TGF-ß1-stimulated fibroblast-mediated collagen gel contraction, migration, and CTHRC1 release in lung fibrotic fibroblasts. Furthermore, pirfenidone attenuated TGF-ß1- and CTHRC1-induced fibroblast activity, upregulation of bone morphogenic protein-4(BMP-4)/Gremlin1, and downregulation of α-smooth muscle actin, fibronectin, and FHL2, similar to that observed post-CTHRC1 inhibition. In contrast, FHL2 inhibition suppressed migration and fibronectin expression, but did not downregulate CTHRC1. CONCLUSIONS: Overall, pirfenidone suppressed fibrotic fibroblast-mediated fibrotic processes via inverse regulation of CTHRC1-induced lung fibroblast activity. Thus, CTHRC1 can be used for predicting pirfenidone response and developing new therapeutic targets for lung fibrosis.

Ac-SDKP increases α-TAT 1 and promotes the apoptosis in lung fibroblasts and epithelial cells double-stimulated with TGF-ß1 and silica.

Crystalline silica (SiO2) particles have very strong toxicity to the lungs, and silicosis is an excessive pulmonary interstitial remodeling disease that follows persistent SiO2 injury. We showed here that DNA double strand breaks (DSBs) and apoptosis were aggravated during rat silicosis induced by SiO2 exposure. Ac-SDKP attenuates lung parenchymal distortion and collagen deposition, and decreases the expression of γH2AX, p21, and cleaved caspase-3, as well as improves the reduction of pulmonary function caused by silicosis. In vitro, we found an evolution of smooth muscle actin α (α-SMA), collagen type I (Col I) in both A549 and MRC-5 cells in response to transforming growth factor-beta 1 (TGF-ß1) + SiO2. Only A549 cells showed any reduction in the rate of apoptosis induced by the double stimulation, because of the anti-apoptotic effects of TGF-ß1. N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is an anti-fibrotic tetrapeptide. It also has the ability to promote the apoptosis of leukemia cells. However its role in promoting cell apoptosis in silicosis is still unknown. We here found that Ac-SDKP could induce cell apoptosis and inhibit fibrotic response in A549 and MRC-5 cells treated with TGF-ß1 + SiO2, and these effects depended on regulation of α-tubulin acetyltransferase 1 (α-TAT1). These findings suggest that Ac-SDKP may have therapeutic value in the treatment of silicotic fibrosis.

Chebulagic acid and Chebulinic acid inhibit TGF-ß1 induced fibrotic changes in the chorio-retinal endothelial cells by inhibiting ERK phosphorylation.

PURPOSE: Diabetic retinopathy (DR) is characterized by pro-inflammatory, pro-angiogenic and pro-fibrotic environment during the various stages of the disease progression. Basement membrane changes in the retina and formation of fibrovascular membrane are characteristically seen in DR. In the present study the effect of Alcoholic (AlE) extracts of Triphala an ayurvedic herbal formulation and its chief compounds, Chebulagic (CA), Chebulinic (CI) and Gallic acid (GA) were evaluated for TGFß1-induced anti-fibrotic activity in choroid-retinal endothelial cells (RF/6A). METHOD: RF/6A cells were treated with TGFß1 alone or co-treated with AlE, CA, CI or GA. The mRNA and protein expression of fibrotic markers (αSMA, CTGF) were assessed by qPCR and western blot/ELISA. Functional changes were assessed using proliferation assay and migration assay. To deduce the mechanism of action, downstream signaling was assessed by western blot analysis along with in silico docking studies. RESULT: AlE (50 µg/ml) CA and CI at 10 µM reduced the expression of pro-fibrotic genes (αSMA and CTGF) induced by TGFß1, by inhibiting ERK phosphorylation. GA did not inhibit TGFß1 mediated changes in RF/6A cells. In silico experiments shows that CA and CI has favourable binding energy to bind with TGFß receptor and inhibit the downstream signaling, while GA did not. CONCLUSION: Hence this study identifies Triphala and its chief compounds CA and CI as potential adjuvants in the management of DR.

Baicalein represses TGF-ß1-induced fibroblast differentiation through the inhibition of miR-21.

Fibroblast-to-myofibroblast differentiation is a highly important pathological characteristic of pulmonary fibrosis. In this study, we aimed to investigate the effects and mechanisms of baicalein on the differentiation of human lung fibroblasts. Baicalein reduced the levels of α-smooth muscle actin (α-SMA) mRNA and protein expression in TGF-ß1-treated human lung fibroblasts. It also decreased the contents of collagen type I and fibronectin in time- and dose-dependent manners, and retarded TGF-ß1-stimulated α-SMA filament formation. Baicalein diminished the expression of miR-21, and miR-21 mimics partially antagonized the effects of baicalein. Additionally, Baicalein inhibited the miR-21 transcriptor STAT3 activity but not AP-1 activity. Moreover, the expression of Spry 1 protein, a miR-21 known target, was improved by baicalein treatment, but the level of Smurf2 protein, another miR-21 target, was not interfered. Collectively, these results demonstrated that baicalein can attenuate TGF-ß1-induced human lung fibroblast differentiation by inhibiting the miR-21 expression.

Simvastatin reduces TGF-ß1-induced SMAD2/3-dependent human ventricular fibroblasts differentiation: Role of protein phosphatase activation.

BACKGROUND: Excessive cardiac fibrosis due to maladaptive remodeling leads to progression of cardiac dysfunction and is modulated by TGF-ß1-activated intracellular phospho-SMAD signaling effectors and transcription regulators. SMAD2/3 phosphorylation, regulated by protein-phosphatases, has been studied in different cell types, but its role in human ventricular fibroblasts (hVFs) is not defined as a target to reduce cytokine-mediated excessive fibrotic response and adverse cardiac remodeling. Statins are a class of drugs reported to reduce cardiac fibrosis, although underlying mechanisms are not completely understood. We aimed to assess whether simvastatin-mediated reduction in TGF-ß1-augmented profibrotic response involves reduction in phospho-SMAD2/3 owing to activation of protein-phosphatase in hVFs. METHODS AND RESULTS: Cultures of hVFs were used. Effect of simvastatin on TGF-ß1-treated hVF proliferation, cytotoxicity, myofibroblast differentiation/activation, profibrotic gene expression and protein-phosphatase activity was assessed. Simvastatin (1 µM) reduced effect of TGF-ß1 (5 ng/mL) on hVF proliferation, myofibroblast differentiation (reduced α-smooth muscle actin [α-SMA-expression]) and activation (decreased procollagen-peptide release). Simvastatin also reduced TGF-ß1-stimulated time-dependent increases in SMAD2/3 phosphorylation and nuclear translocation, mediated through catalytic activation of protein-phosphatases PPM1A and PP2A, which physically interact with SMAD2/3, thereby promoting their dephosphorylation. Effect of simvastatin on TGF-ß1-induced fibroblast activation was annulled by okadaic acid, an inhibitor of protein-phosphatase. CONCLUSIONS: This proof-of-concept study using an in vitro experimental cell culture model identifies the protective role of simvastatin against TGF-ß1-induced hVF transformation into activated myofibroblasts through activation of protein phosphatase, a novel target that can be therapeutically modulated to curb excessive cardiac fibrosis associated with maladaptive cardiac remodeling.