MAPK phosphatase 1 inhibition of p38α within lung myofibroblasts is essential for spontaneous fibrosis resolution.

Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis - effects determined to be mainly dependent on MKP1's dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.

TRIM proteins in fibrosis.

Fibrosis is an outcome of tissue repair after different types of injuries. The homeostasis of extracellular matrix is broken, and excessive deposition occurs, affecting the normal function of tissues and organs, which could become prostrated in serious cases.Finding a suitable target to regulate the repair process and reduce the damage caused by fibrosis is a hot research topic at present. The TRIM family is number of one of the E3 ubiquitin ligase subfamilies and participates in various biological processes including intracellular signal transduction, apoptosis, autophagy, and immunity by regulating the ubiquitination of target proteins. For the past few years, the important role of TRIM in the occurrence and development of fibrosis has been gradually revealed. In this review, we focus on the recent emerging topics on TRIM proteins in the regulation of fibrosis, fibrosis-related cytokines and pathways.

Arsenic-induced lung inflammation and fibrosis in a rat model: Contribution of the HMGB1/RAGE, PI3K/AKT, and TGF-ß1/SMAD pathways.

An increasing number of studies have shown that arsenic exposure increases the risk of lung cancer as well as a variety of non-malignant respiratory diseases, including bronchitis and tracheobronchitis. HMGB1 is widely expressed in a variety of tissues and cells and is involved in the pathological processes of many lung diseases through binding to the corresponding receptors and activating the downstream signaling pathways. However, the exact role of HMGB1/RAGE in arsenic-induced lung injury remains unknown. The aim of this study was to investigate whether HMGB1/RAGE and its activated downstream pathways are involved in the process of arsenic exposure-induced lung injury in rats. In this study, an animal model of oral exposure to arsenic was induced using 2.5, 5 and 10 mg/kg NaAsO2. The results showed that capillary permeability (LDH, TP, ACP, and AKP) was increased in the arsenic exposure groups, resulting in cell damage; this was accompanied by acute inflammation marked by significant neutrophil infiltration. Meanwhile, obvious histopathological damage, including thickening of the lung epithelium, increased infiltration of inflammatory cells, rupture of the alveolar wall, swelling of the mitochondria, and chromatin agglutination was observed by H&E staining and transmission electron microscopy. Furthermore, the results confirmed that the expressions of HMGB1 and RAGE in lung tissue were enhanced, and protein expression of PI3K, p-AKT, IL-1ß, IL-18, and MMP-9 was increased in lung homogenates from the arsenic-exposed groups compared to the control group. Finally, Masson's staining results revealed arsenic-induced fibrosis and collagen deposition. Moreover, a significant increase in key fibrosis factors, including TGF-ß1, p-SMAD2, p-SMAD3, and SMAD4 was observed in the lung homogenates in arsenic-exposed groups. In conclusion, the current study demonstrates that sub-chronic arsenic exposure triggers the inflammatory response and collagen fiber deposition in rat lung tissue. The potential mechanism may be closely related to activation of the pro-inflammatory-related HMGB1/RAGE pathway and initiation of the PI3K/AKT and TGF-ß1/SMAD pathways.

Cystamine Treatment Fails to Prevent the Development of Pulmonary Hypertension in Chronic Hypoxic Rats.

INTRODUCTION: Pulmonary hypertension is characterized by vasoconstriction and remodeling of pulmonary arteries, leading to right ventricular hypertrophy and failure. We have previously found upregulation of transglutaminase 2 (TG2) in the right ventricle of chronic hypoxic rats. The hypothesis of the present study was that treatment with the transglutaminase inhibitor, cystamine, would inhibit the development of pulmonary arterial remodeling, pulmonary hypertension, and right ventricular hypertrophy. METHODS: Effect of cystamine on transamidase activity was investigated in tissue homogenates. Wistar rats were exposed to chronic hypoxia and treated with vehicle, cystamine (40 mg/kg/day in mini-osmotic pumps), sildenafil (25 mg/kg/day), or the combination for 2 weeks. RESULTS: Cystamine concentration-dependently inhibited TG2 transamidase activity in liver and lung homogenates. In contrast to cystamine, sildenafil reduced right ventricular systolic pressure and hypertrophy and decreased pulmonary vascular resistance and muscularization in chronic hypoxic rats. Fibrosis in the lung tissue decreased in chronic hypoxic rats treated with cystamine. TG2 expression was similar in the right ventricle and lung tissue of drug and vehicle-treated hypoxic rats. DISCUSSION/CONCLUSIONS: Cystamine inhibited TG2 transamidase activity, but cystamine failed to prevent pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial muscularization in the chronic hypoxic rat.

Genetic Deletion of Polo-Like Kinase 2 Induces a Pro-Fibrotic Pulmonary Phenotype.

Pulmonary fibrosis is the chronic-progressive replacement of healthy lung tissue by extracellular matrix, leading to the destruction of the alveolar architecture and ultimately death. Due to limited pathophysiological knowledge, causal therapies are still missing and consequently the prognosis is poor. Thus, there is an urgent clinical need for models to derive effective therapies. Polo-like kinase 2 (PLK2) is an emerging regulator of fibroblast function and fibrosis. We found a significant downregulation of PLK2 in four different entities of human pulmonary fibrosis. Therefore, we characterized the pulmonary phenotype of PLK2 knockout (KO) mice. Isolated pulmonary PLK2 KO fibroblasts displayed a pronounced myofibroblast phenotype reflected by increased expression of αSMA, reduced proliferation rates and enhanced ERK1/2 and SMAD2/3 phosphorylation. In PLK2 KO, the expression of the fibrotic cytokines osteopontin and IL18 was elevated compared to controls. Histological analysis of PLK2 KO lungs revealed early stage remodeling in terms of alveolar wall thickening, increased alveolar collagen deposition and myofibroblast foci. Our results prompt further investigation of PLK2 function in pulmonary fibrosis and suggest that the PLK2 KO model displays a genetic predisposition towards pulmonary fibrosis, which could be leveraged in future research on this topic.

Lysyl oxidase directly contributes to extracellular matrix production and fibrosis in systemic sclerosis.

Pulmonary fibrosis is one of the important causes of morbidity and mortality in fibroproliferative disorders such as systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Lysyl oxidase (LOX) is a copper-dependent amine oxidase whose primary function is the covalent crosslinking of collagens in the extracellular matrix (ECM). We investigated the role of LOX in the pathophysiology of SSc. LOX mRNA and protein levels were increased in lung fibroblasts of SSc patients compared with healthy controls and IPF patients. In vivo, bleomycin induced LOX mRNA expression in lung tissues, and LOX activity increased in the circulation of mice with pulmonary fibrosis, suggesting that circulating LOX parallels levels in lung tissues. Circulating levels of LOX were reduced upon amelioration of fibrosis with an antifibrotic peptide. LOX induced ECM production at the transcriptional level in lung fibroblasts, human lungs, and human skin maintained in organ culture. In vivo, LOX synergistically exacerbated fibrosis in bleomycin-treated mice. Further, LOX increased the production of interleukin (IL)-6, and the increase was mediated by LOX-induced c-Fos expression, the nuclear localization of c-Fos, and its engagement with the IL-6 promoter region. Our findings demonstrate that LOX expression and activity correlate with fibrosis in vitro, ex vivo, and in vivo. LOX induced ECM production via upregulation of IL-6 and nuclear localization of c-Fos. Thus, LOX has a direct pathogenic role in SSc-associated fibrosis that is independent of its crosslinking function. Our findings also suggest that measuring circulating LOX levels and activity can be used for monitoring response to antifibrotic therapy.

Significance of nuclear LOXL2 inhibition in fibroblasts and myofibroblasts in the fibrotic process of acute respiratory distress syndrome.

Fibrotic scarring is an important prognostic factor of acute respiratory distress syndrome (ARDS). There are currently no antifibrotic drugs or other therapeutic agents for ARDS. Lysyl oxidase-like 2 (LOXL2), an amine oxidase, contributes to fibrotic scarring by facilitating collagen cross-linking. Recent clinical trials revealed that a monoclonal inhibitory antibody against LOXL2 failed to show benefit over placebo in patients with fibrotic disorders involving the lungs. These clinical results raise the possibility that targeting the extracellular enzymic activity of LOXL2 is not in itself sufficient to prevent fibrotic scarring. We investigated the role of LOXL2 in the pathogenesis of ARDS in vivo, in vitro, and in samples from patients with ARDS. After lung injury, LOXL2 was unevenly expressed in the nuclei of lung fibroblasts and myofibroblasts in the fibrotic phase. Nuclear LOXL2 expression was upregulated in lung fibroblasts after transforming growth factor-beta1 (TGF-ß1)-treatment. LOXL2 silencing abrogated the TGF-ß1-induced expression of a myofibrogenic-progenitor marker, the appearance of proto-myofibroblasts, and the evolution of differentiated myofibroblasts in lung fibroblasts. Nuclear upregulation of Snail was evident in myofibroblasts during the fibrotic phase after lung injury. We detected high levels of LOXL2 protein in the lungs of ARDS patients, specifically during the proliferative and fibrotic phases. Our results highlight nuclear LOXL2 in fibroblasts as a primary causative driver of cell-fate decision toward myofibroblasts and of the progression of fibrotic scarring. A nuclear-LOXL2-targeted agent could be a promising therapeutic strategy against fibrotic disorders including ARDS.

COVID-19 lockdown leads to changes in alcohol consumption patterns. Results from the Polish national survey.

The objective of this study was to investigate the effects of nationwide lockdown during the Novel Coronavirus Disease 2019 (COVID-19) pandemic on an average volume of alcohol consumption and drinking patterns. A survey was conducted with a random sample of 4072 people. The authors found a significant influence of the pandemic period on alcohol consumption compared to the pre-pandemic period. The vast majority of respondents reduced the frequency of consumption of all types of alcohol. However, when the population was divided into subgroups, this differentiation demonstrated that particular groups are more vulnerable to alcohol misuse. Higher frequency of alcohol consumption during the COVID-19 pandemic lockdown was most often found in the group of men, people aged 18-24 years, inhabitants of big cities, and remote workers. Besides, significant differences were observed in subpopulations concerning different types of alcohol. Results emphasized the importance of monitoring and implementation of actions aimed at reducing the potential psychosocial impact of COVID-19, including alcohol-related disorders.

Serpina3n is closely associated with fibrotic procession and knockdown ameliorates bleomycin-induced pulmonary fibrosis.

OBJECTIVE: Pulmonary fibrosis is a fatal interstitial lung disease that is characterized by excessive accumulation of extracellular matrix (ECM) and remodeling of lung. The precise mechanisms underlying pulmonary fibrosis still remain unclear. In the current study, we aimed to investigate the alteration and function of serine (or cysteine) peptidase inhibitor, clade A, member 3 N (Serpina3n) in pulmonary fibrotic models and explore the potential mechanisms. METHODS: We induced pulmonary fibrosis in mice by silica and bleomycin respectively and determined Serpina3n in lung tissues, and then verified the expression of Serpina3n and its correlation with pulmonary fibrosis at seven time points in a bleomycin longstanding model. Moreover, adeno-associated virus type 9 (AAV9)-mediated Serpina3n knockdown was used to treat pulmonary fibrosis in the bleomycin model, whose possible mechanisms would be preliminarily explored by detecting chymotrypsin C as an example. RESULTS: Serpina3n was up-regulated significantly in lungs of both models at mRNA and protein levels relative to control. Notably, the expression of Serpina3n peaked during the 3rd week and then decreased until nearly normal levels during the 10th week, which was closely related to fibrotic procession in bleomycin-treated mice. AAV-mediated Serpina3n knockdown in the lung tissues alleviated bleomycin-induced fibrotic symptoms at various levels and disinhibit chymotrypsin C. CONCLUSIONS: Our study revealed that Serpina3n is a critical regulator in pulmonary fibrosis and suggested Serpina3n inhibition as a potential therapeutic strategy in chronic pulmonary injuries.

Atractylenolide III attenuates bleomycin-induced experimental pulmonary fibrosis and oxidative stress in rat model via Nrf2/NQO1/HO-1 pathway activation.

BACKGROUND: Bleomycin (BLM) is a chemotherapy drug used to treat cancer, one of which side effects is that it can lead to pulmonary fibrosis (PF). Atractylenoide III (AtrIII), derived from the dried roots of rhizoma atractylodis of compositae, is one of the main active substances of rhizoma atractylodis. It has anti-inflammatory, anti-tumor and other effects. This study aimed to investigate whether AtrIII alleviated BLM-induced PF and oxidative stress in rats through the nuclear factor erythroid-2-related factor 2/NQO1,NAD(P)H:quinine oxidoreductase 1/Heme oxygenase-1 (Nrf2/NQO1/HO-1) pathway. METHODS: A BLM-induced pulmonary fibrosis model in SD rats was established. The respiratory dynamics were evaluated by using Wholebody flow-through plethysmography. Lung injury and pulmonary fibrosis were observed by Hematoxylin-eosin (HE) and Masson staining. Apoptosis was assay by Tunel assay. Inflammatory factors were detected with commercial kits. Expression of mRNAs and proteins were detected by RT-qPCR and Western blot, respectively. RESULTS: AtrIII (1.2, 2.4 mg/kg) improved the lung injury and lung function in the BLM-induced Sprague-Dawley (SD) rats. AtrIII reduced the apoptosis rate and protein expression of Caspase-3 and Caspase-9. AtrIII (1.2, 2.4 mg/kg) decrease the pulmonary fibrosis damage and protein expression transforming growth factor-ß (TGF-ß) and α-smooth muscle actin (α-SMA). AtrIII also down-regulated the levels of interleukin 6 (IL-6), inductible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α), while up-regulated the level of IL-10 in peripheral blood serum. Moreover, AtrIII (1.2, 2.4 mg/kg) increased the activity of superoxide dismutase (SOD) and glutathione (GSH), while decreased the malondialdehyde (MDA) content and lactate dehydrogenase (LDH) activity. AtrIII (1.2, 2.4 mg/kg) increased the levels of Nrf2, NQO1 and HO-1. In addition, AtrIII reversed the effects of Nrf2 interference on pulmonary fibrosis damage, decreased SOD and GSH activity, and increased MDA content. CONCLUSION: AtrIII could attenuate the pulmonary fibrosis and reliev oxidative stress through the Nrf2/NQO1/ HO-1 pathway.

Roxadustat attenuates experimental pulmonary fibrosis in vitro and in vivo.

Roxadustat is the first orally administered, small-molecule hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor that has been submitted for FDA regulatory approval to treat anemia secondary to chronic kidney diseases. Its usage has also been suggested for pulmonary fibrosis; however, the corresponding therapeutic effects remain to be investigated. The in vitro effects of roxadustat on cobalt chloride (CoCl2)-stimulated pulmonary fibrosis with L929 mouse fibroblasts as well as on an in vivo pulmonary fibrosismice model induced with bleomycin (BLM; intraperitoneal injection, 50 mg/kg twice a week for 4 continuous weeks) were investigated. It found that the proliferation of L929 cells was inhibited and the production of collagen I, collagen III, prolyl hydroxylase domain protein 2 (PHD2), HIF-1α, α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), transforming growth factor-ß1 (TGF-ß1) and p-Smad3 were reduced relative to that in the CoCl2 or BLM group after roxadustat treatment. Roxadustat ameliorated pulmonary fibrosis by reducing the pathology score and collagen deposition as well as decreasing the expression of collagen I, collagen III, PHD2, HIF-1α, α-SMA, CTGF, TGF-ß1 and p-Smad3/Smad3. Our cumulative results demonstrate that roxadustat administration can attenuate experimental pulmonary fibrosis via the inhibition of TGF-ß1/Smad activation.

Zingerone ameliorates oxidative stress and inflammation in bleomycin-induced pulmonary fibrosis: modulation of the expression of TGF-ß1 and iNOS.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with limited treatment options. Zingerone found in ginger (Zingiber officinale L.) has many pharmacological effects, especially antiinflammatory and antioxidant activity. However, the effect of zingerone on pulmonary fibrosis (PF) is not fully known. The aim of this study was to investigate the effect of zingerone on bleomycin (BLM)-induced PF and its underlying mechanisms. Wistar-albino rats were given single dose of BLM (5 mg/kg, intratracheal) or vehicle (saline). In treatment groups, zingerone (50 and 100 mg/kg, p.o.) was administered orally for 14 days after BLM administration. Rats and lung tissue were weighed to determine lung index. Antioxidant, antiinflammatory effects, and hydroxyproline content of zingerone were determined by ELISA method. Pulmonary inflammation, collagen deposition, and fibrosis score were determined with Hematoxylin-Eosin (HxE) and Masson's trichrome staining. Transforming growth factor-beta 1 (TGF-ß1) and inducible nitric oxide synthase (iNOS) expressions were detected immunohistochemically. BLM administration increased lipid peroxidation (MDA) and decreased superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity. In addition, BLM caused increased levels of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) in bronchoalveolar lavage fluid (BALF) and accumulation of collagen bundles. Zingerone administration decreased collagen accumulation, TNF-α and IL-1ß levels, MDA level, TGF-ß1, and iNOS expression and increased SOD and GPx activity. Histopathological findings supported the results. These results show that zingerone (50 and 100 mg/kg) at both doses significantly contributes to healing of PF by improving inflammation, oxidative stress, and histopathological alterations and by affecting TGF-ß1 and iNOS signaling pathways.

Protective effects of curcumin on bleomycin-induced changes in lung glycoproteins.

The present study investigated the therapeutic effect of curcumin on bleomycin (BLM)-induced alterations in glycoprotein components in the fibrotic lungs. Analysis of the bronchoalveolar lavage fluid (BALF) demonstrated increased fibronectin content at 3, 5, 7, and 14 days after BLM administration. Similarly, lung tissue fibronectin content revealed a progressive increase at various times (days 3, 5, 7, 14, and 28) during the development of lung fibrosis. In addition, alveolar macrophage release of fibronectin was also elevated in BLM-treated rats. Analysis of carbohydrate moieties of glycoproteins revealed an increase in total hexose, fucose, sialic acid and hexosamine levels at 7, 14, and 28 days after BLM treatment. Furthermore, the activities of lung glycosidases such as N-acetyl-ß-D-glucosaminidase, ß-glucosidase, ß-galactosidase, and ß-fucosidase in the fibrotic rats were elevated. Importantly, curcumin significantly inhibited the BLM-induced increases in BALF and lung fibronectin levels. Treatment of BLM rats with curcumin dramatically suppressed alveolar macrophage release of fibronectin. Curcumin also inhibited the increases in complex carbohydrates and glycosidases in the fibrotic lungs. These findings suggest that BLM-induced lung fibrosis is associated with accumulation of glycoproteins, and curcumin has the ability to suppress the enhanced deposition of glycoproteins in the fibrotic lung.

The antifibrotic effect and mechanism of a novel tyrosine kinase inhibitor, ZSP1603, in preclinical models of pulmonary fibrosis.

OBJECTIVE: This study aimed to investigate the efficacy and molecular mechanisms of ZSP1603 as a novel anti-fibrotic compound. MATERIALS AND METHODS: The unilateral left pulmonary fibrosis model was established in the Sprague Dawley (SD) rats. The bilateral pulmonary fibrosis model was established in the C57BL/6J mice. The therapeutic treatment regimen began after the induction of pulmonary fibrosis. The preventive treatment regimen began on the first day of bleomycin administration. Animals were randomly divided into the sham, model, Nintedanib, and ZSP1603 treatment groups. Haematoxylin and eosin (H&E) and Masson's trichrome staining were performed to evaluate pulmonary injury, inflammation, and fibrosis. Cell Counting Kit-8 (CCK-8) assay and Western blot were used to investigate the effects and mechanisms of ZSP1603 on the proliferation of primary human pulmonary fibroblasts (pHPFs). The messenger ribonucleic acid (mRNA) expression of transforming growth factor (TGF)-ß1, tissue inhibitor of metalloproteinase 1 (TIMP-1), and collagen 1A1 (COL1A1) in pHPFs was detected by quantitative Real Time-Polymerase Chain Reaction (PCR). RESULTS: ZSP1603 inhibited the proliferation of pHPFs in vitro by blocking the platelet-derived growth factor receptor-ß (PDGF-Rß) and extracellular signal-regulated kinase (ERK) signalling pathway. ZSP1603 also inhibited the differentiation of pHPFs by reducing the expression of TGF-ß1, TIMP-1, and COL1A1. ZSP1603 significantly attenuated pulmonary injury, inflammation, and fibrosis in vivo in four independent animal studies of pulmonary fibrosis. CONCLUSIONS: ZSP1603 is an effective anti-fibrotic compound with clear mechanisms.

TGF-ß1 increases sialidase 3 expression in human lung epithelial cells by decreasing its degradation and upregulating its translation.

Purpose: We previously found extensive desialylation of glycoconjugates and upregulation of the sialidase enzyme NEU3 in fibrotic lesions in human and mouse lungs. However, studies using microarray analysis of whole lung tissue mRNA and single cell RNA-seq found no significant difference in levels of NEU3 mRNA between IPF patients and controls. This study aimed to elucidate how NEU3 was upregulated in fibrotic lungs.Materials and methods: Transforming growth factor-ß1 (TGF-ß1), a key driver of fibrosis, was added to A549 human alveolar basal epithelial adenocarcinoma cells and human small airway epithelial cells (HSAEpC). NEU3 expression in A549 cells and HSAEpC was detected by immunofluorescence staining. NEU3 translation and degradation were assessed by polysome profiling (polysomes efficiently translate mRNAs; monosomes poorly translate mRNAs) and cycloheximide chase after treating cells with or without TGF-ß1 for 48 h.Results: TGF-ß1 increased NEU3 expression and secretion in A549 cells and HSAEpC but did not change total (nuclear + cytosolic) NEU3 mRNA levels. TGF-ß1 decreased the degradation rate of NEU3 in A549 cells. TGF-ß1 decreased NEU3 mRNA levels in monosomes and increased NEU3 mRNA level in polysomes.Conclusion: TGF-ß1 upregulates levels of NEU3 in epithelial cells by both decreasing NEU3 degradation and by increasing the translation of NEU3 mRNA, explaining the apparent paradox of high levels of NEU3 protein in pulmonary fibrosis without a concomitant increase in the expression of NEU3 mRNA.

Anlotinib attenuated bleomycin-induced pulmonary fibrosis via the TGF-ß1 signalling pathway.

OBJECTIVES: Anlotinib hydrochloride (AL3818) is a novel multitarget tyrosine kinase inhibitor which has the same targets as nintedanib, an effective drug has been approved for the treatment of idiopathic pulmonary fibrosis. Here, we examined whether anlotinib could also attenuate bleomycin-induced pulmonary fibrosis in mice and explored the antifibrosis mechanism. METHODS: We have evaluated the effect of anlotinib on bleomycin-induced pulmonary fibrosis in mice. Inflammatory cytokines in alveolar lavage fluid including IL-1ß, IL-4, IL-6 and TNF-α were determined by ELISA. Biomarkers of oxidative stress were measured by corresponding kit. Histopathologic examination was analysed by H&E staining and immunohistochemistry. In vitro, we investigated whether anlotinib inhibited TGFß/Smad3 and non-Smad pathways by luciferase assay or Western blotting. We also evaluated whether anlotinib inhibited TGF-ß1-induced epithelial-mesenchymal transition (EMT) and promoted myofibroblast apoptosis in order to explore the possible molecular mechanism. KEY FINDINGS: The results indicated that anlotinib treatment remarkably attenuated inflammation, oxidative stress and pulmonary fibrosis in mouse lungs. Anlotinib could inhibit the TGF-ß1 signalling pathway. Additionally, anlotinib not only profoundly inhibited TGF-ß1-induced EMT in alveolar epithelial cells, but also simultaneously reduced the proliferation and promoted the apoptosis in fibroblasts. CONCLUSIONS: In summary, the results suggest that anlotinib-mediated suppression of pulmonary fibrosis is related to the inhibition of TGF-ß1 signalling pathway.

Agonist-specific desensitization of PGE2-stimulated cAMP signaling due to upregulated phosphodiesterase expression in human lung fibroblasts.

Pulmonary fibrosis is characterized by fibroblasts persisting in an activated form, producing excessive fibrous material that destroys alveolar structure. The second messenger molecule cyclic 3',5'-adenosine monophosphate (cAMP) has antifibrotic properties, and prostaglandin E2 (PGE2) can stimulate cAMP production through prostaglandin E (EP)2 and EP4 receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2 have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE2, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE2 or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2 were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2 shifted the concentration-response curve to PGE2 rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2 responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP2- and EP4-selective agonists and antagonists suggested that PGE2-stimulated cAMP responses in HFL-1 cells are mediated by EP2 receptors. EP2 receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2 pretreatment. PGE2 treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE2-mediated desensitization and PDE4 activity was increased, but rolipram did not alter responses to isoproterenol. The PDE3 inhibitor cilostazol had minimal effect. These results show that long-term exposure to PGE2 causes agonist-specific desensitization of EP2 receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family.

Glycogen Synthase Kinase-3ß Inhibition with 9-ING-41 Attenuates the Progression of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with a median survival of 3 years after diagnosis. Although the etiology of IPF is unknown, it is characterized by extensive alveolar epithelial cell apoptosis and proliferation of myofibroblasts in the lungs. While the origins of these myofibroblast appear to be diverse, fibroblast differentiation contributes to expansion of myofibroblasts and to disease progression. We found that agents that contribute to neomatrix formation and remodeling in pulmonary fibrosis (PF); TGF-ß, Factor Xa, thrombin, plasmin and uPA all induced fibroblast/myofibroblast differentiation. These same mediators enhanced GSK-3ß activation via phosphorylation of tyrosine-216 (p-Y216). Inhibition of GSK-3ß signaling with the novel inhibitor 9-ING-41 blocked the induction of myofibroblast markers; α-SMA and Col-1 and reduced morphological changes of myofibroblast differentiation. In in vivo studies, the progression of TGF-ß and bleomycin mediated PF was significantly attenuated by 9-ING-41 administered at 7 and 14 days respectively after the establishment of injury. Specifically, 9-ING-41 treatment significantly improved lung function (compliance and lung volumes; p < 0.05) of TGF-ß adenovirus treated mice compared to controls. Similar results were found in mice with bleomycin-induced PF. These studies clearly show that activation of the GSK-3ß signaling pathway is critical for the induction of myofibroblast differentiation in lung fibroblasts ex vivo and pulmonary fibrosis in vivo. The results offer a strong premise supporting the continued investigation of the GSK-3ß signaling pathway in the control of fibroblast-myofibroblast differentiation and fibrosing lung injury. These data provide a strong rationale for extension of clinical trials of 9-ING-41 to patients with IPF.

TGFß-induced fibroblast activation requires persistent and targeted HDAC-mediated gene repression.

Tissue fibrosis is a chronic disease driven by persistent fibroblast activation that has recently been linked to epigenetic modifications. Here, we screened a small library of epigenetic small-molecule modulators to identify compounds capable of inhibiting or reversing TGFß-mediated fibroblast activation. We identified pracinostat, an HDAC inhibitor, as a potent attenuator of lung fibroblast activation and confirmed its efficacy in patient-derived fibroblasts isolated from fibrotic lung tissue. Mechanistically, we found that HDAC-dependent transcriptional repression was an early and essential event in TGFß-mediated fibroblast activation. Treatment of lung fibroblasts with pracinostat broadly attenuated TGFß-mediated epigenetic repression and promoted fibroblast quiescence. We confirmed a specific role for HDAC-dependent histone deacetylation in the promoter region of the anti-fibrotic gene PPARGC1A (PGC1α) in response to TGFß stimulation. Finally, we identified HDAC7 as a key factor whose siRNA-mediated knockdown attenuates fibroblast activation without altering global histone acetylation. Together, these results provide novel mechanistic insight into the essential role HDACs play in TGFß-mediated fibroblast activation via targeted gene repression.