SPARC Suppresses Apoptosis of Idiopathic Pulmonary Fibrosis Fibroblasts through Constitutive Activation of ��-Catenin

Idiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the accumulation of scar tissue in the lung interstitium. A hallmark of the disease is areas of injury to type II alveolar epithelial cells with attendant accumulation of fibroblasts in areas called fibroblastic foci. In an effort to better characterize the lung fibroblast phenotype in IPF patients, we isolated fibroblasts from patients with IPF and looked for activation of signaling proteins, which could help explain the exaggerated fibrogenic response in IPF. We found that IPF fibroblasts constitutively expressed increased basal levels of SPARC, plasminogen activator inhibitor-1 (PAI-1), and active β-catenin compared with control cells. Control of basal PAI-1 expression in IPF fibroblasts was regulated by SPARC-mediated activation of Akt, leading to inhibition of glycogen synthase kinase-3β and activation of β-catenin. Additionally, IPF fibroblasts (but not control fibroblasts) were resistant to plasminogen-induced apoptosis and were sensitized to plasminogen-mediated apoptosis by inhibition of SPARC or β-catenin. These findings uncover a newly discovered regulatory pathway in IPF fibroblasts that is characterized by elevated SPARC, giving rise to activated β-catenin, which regulates expression of downstream genes, such as PAI-1, and confers an apoptosis-resistant phenotype. Disruption of this pathway may represent a novel therapeutic target in IPF.     

Inhibition of α-SMA by the Ectodomain of FGFR2c Attenuates Lung Fibrosis

The soluble ectodomain of fibroblast growth factor receptor-IIIc (sFGFR2c) is able to bind to fibroblast growth factor (FGF) ligands and block the activation of the FGF-signaling pathway. In this study, sFGFR2c inhibited lung fibrosis dramatically in vitro and in vivo. The upregulation of α-smooth muscle actin (α-SMA) in fibroblasts by transforming growth factor-β1 (TGF-β1) is an important step in the process of lung fibrosis, in which FGF-2, released by TGF-β1, is involved. sFGFR2c inhibited α-SMA induction by TGF-β1 via both the extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad3 pathways in primary mouse lung fibroblasts and the proliferation of mouse lung fibroblasts. In a mouse model of bleomycin (BLM)-induced lung fibrosis, mice were treated with sFGFR2c from d 3 or d 10 to 31 after BLM administration. Then we used hematoxylin and eosin staining, Masson staining and immunohistochemical staining to evaluate the inhibitory effects of sFGFR2c on lung fibrosis. The treatment with sFGFR2c resulted in significant attenuation of the lung fibrosis score and collagen deposition. The expression levels of α-SMA, p-FGFRs, p-ERK1/2 and p-Smad3 in the lungs of sFGFR2c-treated mice were markedly lower. sFGFR2c may have potential for the treatment of lung fibrosis as an FGF-2 antagonist.     

Can a Place of Origin of the Main Cystic Fibrosis Mutations Be Identified?

The genetic background of the mutations that most often cause cystic fibrosis (CF) is different from that of non-CF chromosomes in populations of European origin. It is not known whether these haplotype backgrounds could be found at high frequencies in populations in which CF is, at present, not common; such populations would be candidates for the place of origin of CF mutations. An analysis of haplotypes of CF transmembrane conductance regulator, together with their variation in specific CF chromosomes, in a worldwide survey of normal chromosomes shows (1) a very low frequency or absence of the most common CF haplotypes in all populations analyzed and (2) a strong genetic variability and divergence, among various populations, of the chromosomes that carry disease-causing mutations. The depth of the gene genealogy associated with disease-causing mutations may be greater than that of the evolutionary process that gave rise to present-day human populations. The concept of "population of origin" lacks either spatial or temporal meaning for mutations that are likely to have been present in Europeans before the ethnogenesis of present populations; subsequent population processes may have erased the traces of their geographic origin.     

Sorafenib Ameliorates Renal Fibrosis through Inhibition of TGF-β-Induced Epithelial-Mesenchymal Transition

ObjectiveThis study was to investigate whether sorafenib can inhibit the progression of renal fibrosis and to study the possible mechanisms of this effect.MethodsEight-week-old rats were subjected to unilateral ureteral obstruction (UUO) and were intragastrically administered sorafenib, while control and sham groups were administered vehicle for 14 or 21 days. NRK-52E cells were treated with TGF-β1 and sorafenib for 24 or 48 hours. HE and Masson staining were used to visualize fibrosis of the renal tissue in each group. The expression of α-SMA and E-cadherin in kidney tissue and NRK-52E cells were performed using immunohistochemistry and immunofluorescence. The apoptosis rate of NRK-52E cells was determined by flow cytometry analysis. The protein levels of Smad3 and p-Smad3 in kidney tissue and NRK-52E cells were detected by western blot analysis.ResultsHE staining demonstrated that kidney interstitial fibrosis, tubular atrophy, and inflammatory cell infiltration in the sorafenib-treated-UUO groups were significantly decreased compared with the vehicle-treated-UUO group (p<0.05). Masson staining showed that the area of fibrosis was significantly decreased in the sorafenib-treated-UUO groups compared with vehicle-treated-UUO group (p<0.01). The size of the kidney did not significantly increase; the cortex of the kidney was thicker and had a richer blood supply in the middle-dose sorafenib group compared with the vehicle-treated-UUO group (p<0.05). Compared with the vehicle-treated-UUO and TGF-β-stimulated NRK-52E groups, the expression of a-SMA and E-cadherin decreased and increased, respectively, in the UUO kidneys and NRK-52E cells of the sorafenib-treated groups (p<0.05). The apoptotic rate of NRK-52E cells treated with sorafenib decreased for 24 hours in a dose-dependent manner (p<0.05). Compared with the vehicle-treated UUO and TGF-β-stimulated NRK-52E groups, the ratio of p-Smad3 to Smad3 decreased in the sorafenib-treated groups (p<0.05).ConclusionOur results suggest that sorafenib may useful for the treatment of renal fibrosis through the suppression of TGF-β/Smad3-induced EMT signaling.     

IFN-γ Stimulates Autophagy-Mediated Clearance of Burkholderia cenocepacia in Human Cystic Fibrosis Macrophages

Burkholderia cenocepacia is a virulent pathogen that causes significant morbidity and mortality in patients with cystic fibrosis (CF), survives intracellularly in macrophages, and uniquely causes systemic infections in CF. Autophagy is a physiologic process that involves engulfing non-functional organelles and proteins and delivering them for lysosomal degradation, but also plays a role in eliminating intracellular pathogens, including B. cenocepacia. Autophagy is defective in CF but can be stimulated in murine CF models leading to increased clearance of B. cenocepacia, but little is known about autophagy stimulation in human CF macrophages. IFN-γ activates macrophages and increases antigen presentation while also inducing autophagy in macrophages. We therefore, hypothesized that treatment with IFN-γ would increase autophagy and macrophage activation in patients with CF. Peripheral blood monocyte derived macrophages (MDMs) were obtained from CF and non-CF donors and subsequently infected with B. cenocepacia. Basal serum levels of IFN-γ were similar between CF and non-CF patients, however after B. cenocepacia infection there is deficient IFN-γ production in CF MDMs. IFN-γ treated CF MDMs demonstrate increased co-localization with the autophagy molecule p62, increased autophagosome formation, and increased trafficking to lysosomes compared to untreated CF MDMs. Electron microscopy confirmed IFN-γ promotes double membrane vacuole formation around bacteria in CF MDMs, while only single membrane vacuoles form in untreated CF cells. Bacterial burden is significantly reduced in autophagy stimulated CF MDMs, comparable to non-CF levels. IL-1β production is decreased in CF MDMs after IFN-γ treatment. Together, these results demonstrate that IFN-γ promotes autophagy-mediated clearance of B. cenocepacia in human CF macrophages.     

Activation of TGF-β Pathway by Areca Nut Constituents: A Possible Cause of Oral Submucous Fibrosis

Oral submucous fibrosis (OSF) is a chronic inflammatory disease characterized by the accumulation of excess collagen, and areca nut chewing has been proposed as an important etiological factor for disease manifestation. Activation of transforming growth factor-β signaling has been postulated as the main causative event for increased collagen production in OSF. Oral epithelium plays important roles in OSF, and arecoline has been shown to induce TGF-β in epithelial cells. In an attempt to understand the role of areca nut constituents in the manifestation of OSF, we studied the global gene expression profile in epithelial cells (HaCaT) following treatment with areca nut water extract or TGF-β. Interestingly, 64% of the differentially regulated genes by areca nut water extract matches with the TGF-β induced gene expression profile. Out of these, expression of 57% of genes was compromised in the presence of ALK5 (TβRI) inhibitor and 7% were independently induced by areca nut, highlighting the importance of TGF-β in areca nut actions. Areca nut water extract treatment induced p-SMAD2 and TGF-β downstream targets in HaCaT cells but not in human gingival fibroblast cells (hGF), suggesting epithelial cells could be the source of TGF-β in promoting OSF. Water extract of areca nut consists of polyphenols and alkaloids. Both polyphenol and alkaloid fractions of areca nut were able to induce TGF-β signaling and its downstream targets. Also, SMAD-2 was phosphorylated following treatment of HaCaT cells by Catechin, Tannin and alkaloids namely Arecoline, Arecaidine and Guvacine. Moreover, both polyphenols and alkaloids induced TGF-β2 and THBS1 (activator of latent TGF-β) in HaCaT cells suggesting areca nut mediated activation of p-SMAD2 involves up-regulation and activation of TGF-β. These data suggest a major causative role for TGF-β that is induced by areca nut in OSF progression.     

Inhibition of Notch Signaling by a γ-Secretase Inhibitor Attenuates Hepatic Fibrosis in Rats

Notch signaling is essential to the regulation of cell differentiation, and aberrant activation of this pathway is implicated in human fibrotic diseases, such as pulmonary, renal, and peritoneal fibrosis. However, the role of Notch signaling in hepatic fibrosis has not been fully investigated. In the present study, we show Notch signaling to be highly activated in a rat model of liver fibrosis induced by carbon tetrachloride (CCl₄), as indicated by increased expression of Jagged1, Notch3, and Hes1. Blocking Notch signaling activation by a γ-secretase inhibitor, DAPT, significantly attenuated liver fibrosis and decreased the expression of snail, vimentin, and TGF-β1 in association with the enhanced expression of E-cadherin. The study in vitro revealed that DAPT treatment could suppress the EMT process of rat hepatic stellate cell line (HSC-T6). Interestingly, DAPT treatment was found not to affect hepatocyte proliferation in vivo. In contrast, DAPT can inhibit hepatocyte apoptosis to some degree. Our study provides the first evidence that Notch signaling is implicated in hepatic fibrogenesis and DAPT treatment has a protective effect on hepatocytes and ameliorates liver fibrosis. These findings suggest that the inhibition of Notch signaling might present a novel therapeutic approach for hepatic fibrosis.     

Role of IL-1β in Experimental Cystic Fibrosis upon P. aeruginosa Infection

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1β in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftr ^tm1eur or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1^−/−), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1^−/− double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1β and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1β signaling in response to P. aeruginosa. IL-1β antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1β antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1β production and reduced bacterial clearance. Further, we show that neutralization of IL-1β in d/d mice through the double mutation d/d x IL-1R1^−/− and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1β pathway may be detrimental in CF patients.     

Attenuation of CCl4-Induced Hepatic Fibrosis in Mice by Vaccinating against TGF-β1

Transforming growth factor β1 (TGF-β1) is the pivotal pro-fibrogenic cytokine in hepatic fibrosis. Reducing the over-produced expression of TGF-β1 or blocking its signaling pathways is considered to be a promising therapeutic strategy for hepatic fibrosis. In this study, we evaluated the feasibility of attenuating hepatic fibrosis by vaccination against TGF-β1 with TGF-β1 kinoids. Two TGF-β1 kinoid vaccines were prepared by cross-linking TGF-β1-derived polypeptides (TGF-β1²⁵–[41-65] and TGF-β1³⁰–[83-112]) to keyhole limpet hemocyanin (KLH). Immunization with the two TGF-β1 kinoids efficiently elicited the production of high-levels of TGF-β1-specific antibodies against in BALB/c mice as tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The antisera neutralized TGF-β1-induced growth-inhibition on mink lung epithelial cells (Mv1Lu) and attenuated TGF-β1-induced Smad2/3 phosphorylation, α-SMA, collagen type 1 alpha 2 (COL1A2), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) expression in the rat hepatic stellate cell (HSC) line, HSC-T6. Vaccination against TGF-β1 with the kinoids significantly suppressed CCl₄-induced collagen deposition and the expression of α-SMA and desmin, attenuated hepatocyte apoptosis and accelerated hepatocyte proliferation in BALB/c mice. These results demonstrated that immunization with the TGF-β1 kinoids efficiently attenuated CCl₄-induced hepatic fibrosis and liver injury. Our study suggests that vaccination against TGF-β1 might be developed into a feasible therapeutic approach for the treatment of chronic fibrotic liver diseases.     

Involvement of the Wnt/β-Catenin Signaling Pathway in the Cellular and Molecular Mechanisms of Fibrosis in Endometriosis

Background

During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified.

Objectives

The objective of the present study was to investigate whether the Wnt/β-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/β-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice.

Methods

Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/β-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/β-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice.

Results

Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-β1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis.

Conclusion

Aberrant activation of the Wnt/β-catenin pathway may be involved in mediating fibrogenesis in endometriosis.     

Targeting Myofibroblasts in Model Systems of Fibrosis by an Artificial α-Smooth Muscle-Actin Promoter Hybrid

Myofibroblasts are the main cell types producing extracellular matrix proteins in a variety of fibrotic diseases. Therefore, they are useful targets for studies of intracellular communication and gene therapeutical approaches in scarring diseases. An artificial promoter containing the −702 bp regulatory sequence of the α-smooth muscle actin (SMA) gene linked to the first intron enhancer sequence of the β-actin gene and the β-globin intron-exon junction was constructed and tested for myofibroblast-dependent gene expression using the green fluorescent protein as a reporter. Reporter expression revealed myofibroblast-specific function in hepatic and renal myofibroblasts, in vitro. In addition, differentiation-dependent activation of the SMA-β-actin promoter hybrid was shown after induction of myofibroblastic features in mesangial cells by stretching treatment. Furthermore, wound healing experiments with SMA-β-actin promoter reporter mice demonstrated myofibroblast-specific action, in vivo. In conclusion, the −702 bp regulatory region of the SMA promoter linked to enhancing β-actin and β-globin sequences benefits from its small size and is suggested as a promising tool to target myofibroblasts as the crucial cell type in various scarring processes.     

Lack of a 5.9 kDa Peptide C-Terminal Fragment of Fibrinogen α Chain Precedes Fibrosis Progression in Patients with Liver Disease

Early detection of fibrosis progression is of major relevance for the diagnosis and management of patients with liver disease. This study was designed to find non-invasive biomarkers for fibrosis in a clinical context where this process occurs rapidly, HCV-positive patients who underwent liver transplantation (LT). We analyzed 93 LT patients with HCV recurrence, 41 non-LT patients with liver disease showing a fibrosis stage F≥1 and 9 patients without HCV recurrence who received antiviral treatment before LT, as control group. Blood obtained from 16 healthy subjects was also analyzed. Serum samples were fractionated by ion exchange chromatography and their proteomic profile was analyzed by SELDI-TOF-MS. Characterization of the peptide of interest was performed by ion chromatography and electrophoresis, followed by tandem mass spectrometry identification. Marked differences were observed between the serum proteome profile of LT patients with early fibrosis recurrence and non-recurrent LT patients. A robust peak intensity located at 5905 m/z was the distinguishing feature of non-recurrent LT patients. However, the same peak was barely detected in recurrent LT patients. Similar results were found when comparing samples of healthy subjects with those of non-LT fibrotic patients, indicating that our findings were not related to either LT or HCV infection. Using tandem mass-spectrometry, we identified the protein peak as a C-terminal fragment of the fibrinogen α chain. Cell culture experiments demonstrated that TGF-β reduces α-fibrinogen mRNA expression and 5905 m/z peak intensity in HepG2 cells, suggesting that TGF-β activity regulates the circulating levels of this protein fragment. In conclusion, we identified a 5.9 kDa C-terminal fragment of the fibrinogen α chain as an early serum biomarker of fibrogenic processes in patients with liver disease.     

Clinical Relevance of Plasma Prostaglandin F2α Metabolite Concentrations in Patients with Idiopathic Pulmonary Fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology with few current treatment options. Recently, we determined an important role of prostaglandin F_2α (PGF_2α) in pulmonary fibrosis by using a bleomycin-induced pulmonary fibrosis model and found an abundance of PGF_2α in bronchoalveolar lavage fluid of IPF patients. We investigated the role of PGF_2α in human IPF by assessing plasma concentrations of 15-keto-dihydro PGF_2α, a stable metabolite of PGF_2α.

Methods

We measured plasma concentrations of 15-keto-dihydro PGF_2α in 91 IPF patients and compared these values with those of controls (n = 25). We further investigated the relationships of plasma 15-keto-dihydro PGF_2α concentrations with disease severity and mortality.

Results

Plasma concentrations of 15-keto-dihydro PGF_2α were significantly higher in IPF patients than controls (p<0.001). Plasma concentrations of this metabolite were significantly correlated with forced expiratory volume in 1 second (Rs [correlation coefficient] = −0.34, p = 0.004), forced vital capacity (Rs = −0.33, p = 0.005), diffusing capacity for carbon monoxide (Rs = −0.36, p = 0.003), the composite physiologic index (Rs = 0.40, p = 0.001), 6-minute walk distance (Rs = −0.24, p = 0.04) and end-exercise oxygen saturation (Rs = −0.25, p = 0.04) when patients with emphysema were excluded. Multivariate analysis using stepwise Cox proportional hazards model showed that a higher composite physiologic index (relative risk = 1.049, p = 0.002) and plasma 15-keto-dihydro PGF_2α concentrations (relative risk = 1.005, p = 0.002) were independently associated with an increased risk of mortality.

Conclusions

We demonstrated significant associations of plasma concentrations of PGF_2α metabolites with disease severity and prognosis, which support a potential pathogenic role for PGF_2α in human IPF.     

Patient-Specific Modeling of Regional Antibiotic Concentration Levels in Airways of Patients with Cystic Fibrosis: Are We Dosing High Enough?

Background

Pseudomonas aeruginosa (Pa) infection is an important contributor to the progression of cystic fibrosis (CF) lung disease. The cornerstone treatment for Pa infection is the use of inhaled antibiotics. However, there is substantial lung disease heterogeneity within and between patients that likely impacts deposition patterns of inhaled antibiotics. Therefore, this may result in airways below the minimal inhibitory concentration of the inhaled agent. Very little is known about antibiotic concentrations in small airways, in particular the effect of structural lung abnormalities. We therefore aimed to develop a patient-specific airway model to predict concentrations of inhaled antibiotics and to study the impact of structural lung changes and breathing profile on local concentrations in airways of patients with CF.

Methods

In- and expiratory CT-scans of children with CF (5–17 years) were scored (CF-CT score), segmented and reconstructed into 3D airway models. Computational fluid dynamic (CFD) simulations were performed on 40 airway models to predict local Aztreonam lysine for inhalation (AZLI) concentrations. Patient-specific lobar flow distribution and nebulization of 75 mg AZLI through a digital Pari eFlow model with mass median aerodynamic diameter range were used at the inlet of the airway model. AZLI concentrations for central and small airways were computed for different breathing patterns and airway surface liquid thicknesses.

Results

In most simulated conditions, concentrations in both central and small airways were well above the minimal inhibitory concentration. However, small airways in more diseased lobes were likely to receive suboptimal AZLI. Structural lung disease and increased tidal volumes, respiratory rates and larger particle sizes greatly reduced small airway concentrations.

Conclusions

CFD modeling showed that concentrations of inhaled antibiotic delivered to the small airways are highly patient specific and vary throughout the bronchial tree. These results suggest that anti-Pa treatment of especially the small airways can be improved.