Upregulation of activin-B and follistatin in pulmonary fibrosis - a translational study using human biopsies and a specific inhibitor in mouse fibrosis models.

BACKGROUND: Activins are members of the TGF-ß superfamily of growth factors. First, we identified by expression array screening that activin-B and follistatin are upregulated in human idiopathic pulmonary fibrosis (IPF). Next, we wanted to clarify their specific role in lung fibrosis formation. METHODS: We used specific antibodies for activin-A and -B subunits and follistatin to measure and localize their levels in idiopathic pulmonary fibrosis and control lung biopsies. To inhibit activin signaling, we used soluble activin type IIB receptor fused to the Fc portion of human IgG1 (sActRIIB-Fc) in two different mouse models of pulmonary fibrosis. RESULTS: Activin-B and follistatin mRNA levels were elevated in the human IPF lung. Immunoreactivity to activin-A, -B and follistatin localized predominantly to the hyperplastic, activated alveolar epithelium, but was also seen in inflammatory cells. Mice treated with sActRIIB-Fc showed increased skeletal muscle mass and a clear reduction in alveolar cell counts in bronchoalveolar lavage fluid, but no significant antifibrotic effect in the lung was observed. CONCLUSIONS: The upregulation of activin-B and follistatin in IPF is a novel finding. Our results indicate that activin inhibition is not an efficient tool for antifibrotic therapy, but could be useful in reducing alveolar cellular response to injury. Activin-B and follistatin levels may be useful as biomarkers of IPF.

Bone morphogenetic protein-inducer tilorone identified by high-throughput screening is antifibrotic in vivo.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with a poor prognosis and very few therapeutic options. On the molecular level, patients with IPF have increased amounts of the bone morphogenetic protein (BMP) inhibitor gremlin in their lungs, which results in decreased BMP signaling, and an increase in transforming growth factor-ß signaling. Based on these findings, we hypothesized that restoration of the impaired BMP signaling would offer a novel strategy for the prevention of fibrosis progression or for the treatment of pulmonary fibrosis. We used reporter cell lines and high-throughput screening of a chemical compound library as an approach to finding molecules that increase BMP signaling in lung epithelial cells, without increasing transforming growth factor-ß signaling. The most promising candidate drug was analyzed further by studying its effects on BMP target gene expression, Smad protein phosphorylation, and a mouse model of silica-induced pulmonary fibrosis. The most promising drug candidate, tilorone, induced BMP signaling in the reporter cells and increased the expression of BMP-7 and a BMP target gene, Id3, in lung epithelial A549 cells. In a mouse model of pulmonary fibrosis, tilorone decreased lung hydroxyproline content and the expression of collagen genes Col1A1 and Col3A1. Mice treated with tilorone showed markedly decreased histological changes, compared with untreated mice. These findings indicate that tilorone has biologically significant antifibrotic properties.

Cysteine-rich protein 1 is regulated by transforming growth factor-ß1 and expressed in lung fibrosis.

Transforming growth factor-ß (TGF-ß) is a diverse cytokine regulating growth, apoptosis, differentiation, adhesion, invasion, and extracellular matrix production. Dysregulation of TGF-ß is associated with fibrotic disorders and epithelial-mesenchymal transition, and has been linked with idiopathic pulmonary fibrosis (IPF). Cysteine-rich protein 1 (CRP1) is a small LIM-domain containing protein involved in smooth muscle differentiation. Here, we show that TGF-ß1 increases the expression of CRP1 protein and that CRP1 levels increase in a biphasic fashion. A rapid transient (15-45 min) increase in CRP1 is followed by a subsequent, sustained increase in CRP1 a few hours afterwards that lasts several days. We find that TGF-ß1 regulates the expression of CRP1 through Smad and non-conventional p38 MAPK signaling pathways in a transcription-independent manner and that the induction occurs concomitant with an increase in myofibroblast differentiation. Using CRP1 silencing by shRNA, we identify CRP1 as a novel factor mediating cell contractility. Furthermore, we localize CRP1 to fibroblastic foci in IPF lungs and find that CRP1 is significantly more expressed in IPF as compared to control lung tissue. The results show that CRP1 is a novel TGF-ß1 regulated protein that is expressed in fibrotic lesions and may be relevant in the IPF disease.

Regulation of TGF-ß storage and activation in the human idiopathic pulmonary fibrosis lung.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of unknown cause. The pathogenesis of the disease is characterized by fibroblast accumulation and excessive transforming growth factor-ß (TGF-ß) activation. Although TGF-ß activation is a complex process involving various protein interactions, little is known of the specific routes of TGF-ß storage and activation in human lung. Here, we have systematically analyzed the expression of specific proteins involved in extracellular matrix targeting and activation of TGF-ß. Latent TGF-ß-binding protein (LTBP)-1 was found to be significantly upregulated in IPF patient lungs. LTBP-1 expression was especially high in the fibroblastic foci, in which P-Smad2 immunoreactivity, indicative of TGF-ß signaling activity, was less prominent. In cultured primary lung fibroblasts and epithelial cells, short-interfering-RNA-mediated downregulation of LTBP-1 resulted in either increased or decreased TGF-ß signaling activity, respectively, suggesting that LTBP-1-mediated TGF-ß activation is dependent on the cellular context in the lung. Furthermore, LTBP-1 was shown to colocalize with fibronectin, fibrillin-1 and fibrillin-2 proteins in the IPF lung. Fibrillin-2, a developmental gene expressed only in blood vessels in normal adult lung, was found specifically upregulated in IPF fibroblastic foci. The TGF-ß-activating integrin ß8 subunit was expressed at low levels in both control and IPF lungs. Alterations in extracellular matrix composition, such as high levels of the TGF-ß storage protein LTBP-1 and the re-appearance of fibrillin-2, probably modulate TGF-ß availability and activation in different pulmonary compartments in the fibrotic lung.

Transcription factor GATA-6 is expressed in quiescent myofibroblasts in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) (histopathology of usual interstitial pneumonia [UIP]) is a progressive disease with poor prognosis. Characteristic features of IPF/UIP include fibroblastic foci, which are patchy lesions of focal, disarranged myofibroblasts. GATA-6 is a transcription factor linked with cell differentiation. Its role in the development of IPF has not previously been investigated. We hypothesized that GATA-6 participates in the differentiation of fibroblasts into myofibroblasts in IPF/UIP lungs. The expression patterns of GATA-6, the mesenchymal marker alpha-smooth muscle actin (alpha-SMA), and markers for proliferation (Ki67) and apoptosis (caspase-3) were analyzed in human IPF/UIP tissue samples. The effects of GATA-6 overexpression and silencing were studied in cell cultures. The results show that the alpha-SMA-positive fibroblastic foci in IPF/UIP lungs are positive for GATA-6, but negative for Ki67 and caspase-3. Cultured human IPF/UIP fibroblasts expressed GATA-6 mRNA, whereas cells from the normal adult lung did not. In cultured A549 lung epithelial cells, the induction of GATA-6 by transforming growth factor-beta1 resulted in simultaneous expression of alpha-SMA and decrease of E-cadherin. The inhibition of GATA-6 expression in fibroblasts showed that GATA-6 mediates the alpha-SMA-inducing signal of transforming growth factor-beta1. In conclusion, the hallmark of IPF/UIP histopathology, the fibroblast focus, consists of differentiated, quiescent cells that prominently express GATA-6.

Characterization of the alnumycin gene cluster reveals unusual gene products for pyran ring formation and dioxan biosynthesis.

Alnumycin is closely related to the benzoisochromanequinone (BIQ) polyketides such as actinorhodin. Exceptional structural features include differences in aglycone tailoring that result in the unique alnumycin chromophore and the existence of an unusual 4-hydroxymethyl-5-hydroxy-1,3-dioxan moiety. Cloning and sequencing of the alnumycin gene cluster from Streptomyces sp. CM020 revealed expected biosynthesis genes for polyketide assembly, but several genes encoding subsequent tailoring enzymes were highly atypical. Heterologous expression studies confirmed that all of the genes required for alnumycin biosynthesis resided within the sequenced clone. Inactivation of genes aln4 and aln5 showed that the mechanism of pyran ring formation differs from actinorhodin and granaticin pathways. Further inactivation studies identified two genes, alnA and alnB, involved in the synthesis and attachment of the dioxan moiety, and resulted in the production of the polyketide prealnumycin.

Reduced phosphorylation of the TGF-Beta signal transducer Smad2 in emphysematous human lung.

Chronic obstructive pulmonary disease (COPD) is characterized by inflammatory immune response, emphysematous destruction of alveolar structures and obstruction in the small conducting airways. Transforming growth factor (TGF)-beta is involved in the maintenance of normal lung tissue homeostasis as a regulator of extracellular proteolysis, tissue repair and inflammatory functions. This study was undertaken to characterize TGF-beta signaling in pathologically distinct areas of COPD lungs. Using Smad2 phosporylation (P-Smad2) as an indicator of TGF-beta signaling activity we analyzed COPD patient tissues and controls by immunohistochemistry. Emphysematous lung showed significantly reduced P-Smad2 immunoreactivity both in the alveolar and bronchiolar epithelium, which is evidence of reduced TGF-beta signaling activity. On the contrary, in the thickened peribronchial areas there was an increase in the amount of P-Smad2 positive cells. Isolated COPD lung fibroblasts also displayed increased TGF-beta signaling and target gene expression suggesting that the fibroblasts are characteristic to the small airway disease phenotype. Our results indicate that TGF-beta signaling activity is differentially regulated in distinct areas of COPD lung and likely contributes to both emphysematous development and small airway obstruction.

Borrelia burgdorferi inhibits human neutrophil functions.

Outer surface proteins OspA and OspB are among the most prominent Borrelia burgdorferi surface molecules. We constructed OspAB and OspA complementation mutants of B. burgdorferi Osp-less strain B313 and investigated the role of these surface proteins in the interactions of B. burgdorferi, human neutrophils and the complement system. We found that (1) OspB inhibits the phagocytosis and oxidative burst of human neutrophils at low serum concentrations, whereas OspA induces the oxidative burst in neutrophils; (2) OspB may have an inhibiting role in serum sensitivity and complement activation; (3) all studied strains inhibit the chemotaxis of human neutrophils specifically towards fMLP but not towards C5a, regardless of their Osp expression. These results suggest that although OspA and OspB are co-ordinately transcribed, they differ in their effects on human neutrophil functions. Our findings suggest that B. burgdorferi exploits a wide variety of immune evasion mechanisms, besides previously documented complement resistance, to survive in the vertebrate host.

Peroxiredoxin II expression and its association with oxidative stress and cell proliferation in human idiopathic pulmonary fibrosis.

Oxidant burden has been suggested to be a contributor to the pathogenesis of idiopathic pulmonary fibrosis (IPF). The study focused on peroxiredoxin (Prx) II, an antioxidant that has been associated with platelet-derived growth factor (PDGF) signaling and consequent cell proliferation. Localization and expression of Prx II, PDGF receptors (PDGFRalpha, PDGFRbeta), Ki67, and nitrotyrosine were assessed in control (n=10) and IPF/usual interstitial pneumonia (UIP) (n=10) lung biopsies by immunohistochemistry and morphometry. Prx II oxidation was determined by standard and non-reducing Western blots, two-dimensional gel electrophoresis, and mass spectrometry. Prx II localized in the IPF/UIP epithelium and alveolar macrophages. Prx II-positive area in the fibroblastic foci (FF) was smaller than in other parenchymal areas (p=0.03) or in the hyperplastic epithelium (p=0.01). There was no major Prx II oxidation in IPF/UIP compared with the normal lung. The FF showed only minor immunoreactivity to the PDGFRs; Ki67, a marker of cell proliferation; and nitrotyrosine, a marker of oxidative/nitrosative stress. The results suggest that Prx II oxidation does not relate to the pathogenesis of IPF/UIP and that Prx II, PDGFRs, and proliferating cells colocalize in the IPF/UIP lung. Unexpectedly, FF represented areas of low cell proliferation.

Antibacterial effects and dissolution behavior of six bioactive glasses.

Dissolution behavior of six bioactive glasses was correlated with the antibacterial effects of the same glasses against sixteen clinically important bacterial species. Powdered glasses (<45 microm) were immersed in simulated body fluid (SBF) for 48 h. The pH in the solution inside the glass powder was measured in situ with a microelectrode. After 2, 4, 27, and 48 h, the pH and concentration of ions after removing the particles and mixing the SBF were measured with a normal glass pH electrode and ICP-OES. The bacteria were cultured in broth with the glass powder for up to 4 days, after which the viability of the bacteria was determined. The antibacterial effect of the glasses increased with increasing pH and concentration of alkali ions and thus with increased dissolution tendency of the glasses, but it also depended on the bacterium type. The changes in the concentrations of Si, Ca, Mg, P, and B ions in SBF did not show statistically significant influence on the antibacterial property. Bioactive glasses showed strong antibacterial effects for a wide selection of aerobic bacteria at a high sample concentration (100 mg/mL). The antibacterial effects increased with glass concentration and a concentration of 50 mg/mL (SA/V 185 cm(-1)) was required to generate the bactericidal effects. Understanding the dissolution mechanisms of bioactive glasses is essential when assessing their antibacterial effects.

Antibacterial effect of bioactive glasses on clinically important anaerobic bacteria in vitro.

Bioactive glasses (BAGs) of different compositions have been studied for decades for clinical use and they have found many dental and orthopaedic applications. Particulate BAGs have also been shown to have antibacterial properties. This large-scale study shows that two bioactive glass powders (S53P4 and 13-93) and a sol-gel derived material (CaPSiO II) have an antibacterial effect on 17 clinically important anaerobic bacterial species. All the materials tested demonstrated growth inhibition, although the concentration and time needed for the effect varied depending on the BAG. Glass S53P4 had a strong growth-inhibitory effect on all pathogens tested. Glass 13-93 and sol-gel derived material CaPSiO II showed moderate antibacterial properties.

Bactericidal effects of bioactive glasses on clinically important aerobic bacteria.

Bioactive glasses (BAGs) have been studied for decades for clinical use, and they have found many dental and orthopedic applications. BAGs have also been shown to have an antibacterial effect e.g., on some oral microorganisms. In this extensive work we show that six powdered BAGs and two sol-gel derived materials have a clear antibacterial effect on 29 clinically important bacterial species. We also incorporated a rapid and accurate flow cytometric (FCM) method to calculate and standardize the numbers of viable bacteria inoculated in the suspensions used in the tests for antibacterial activity. In all materials tested growth inhibition could be demonstrated, although the concentration and time needed for the effect varied depending on the BAG. The most effective glass was S53P4, which had a clear growth-inhibitory effect on all pathogens tested. The sol-gel derived materials CaPSiO and CaPSiO II also showed a strong antibacterial effect. In summary, BAGs were found to clearly inhibit the growth of a wide selection of bacterial species causing e.g., infections on the surfaces of prostheses in the body after implantation.