BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung.Methods and FindingsUsing oligonucleotide arrays, we identified osteopontin as one of the genes that significantly distinguishes IPF from normal lungs. Osteopontin was localized to alveolar epithelial cells in IPF lungs and was also significantly elevated in bronchoalveolar lavage from IPF patients. To study the fibrosis-relevant effects of osteopontin we stimulated primary human lung fibroblasts and alveolar epithelial cells (A549) with recombinant osteopontin. Osteopontin induced a significant increase of migration and proliferation in both fibroblasts and epithelial cells. Epithelial growth was inhibited by the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) and antibody to CD44, while fibroproliferation was inhibited by GRGDS and antibody to αvβ3 integrin. Fibroblast and epithelial cell migration were inhibited by GRGDS, anti-CD44, and anti-αvβ3. In fibroblasts, osteopontin up-regulated tissue inhibitor of metalloprotease-1 and type I collagen, and down-regulated matrix metalloprotease-1 (MMP-1) expression, while in A549 cells it caused up-regulation of MMP-7. In human IPF lungs, osteopontin colocalized with MMP-7 in alveolar epithelial cells, and application of weakest link statistical models to microarray data suggested a significant interaction between osteopontin and MMP-7.ConclusionsOur results provide a potential mechanism by which osteopontin secreted from the alveolar epithelium may exert a profibrotic effect in IPF lungs and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease.

Keywords: ATG4B, autophagy, autophagin-1, epithelial cell, idiopathic pulmonary fibrosis, lung fibrosis Abbreviations: ACTA2, actin, a 2, smooth muscle, aorta; ATG3, autophagy-related 3; ATG4B, autophagy-related 4B; cysteine peptidase; ATG5, autophagy-related 5; ATG7, autophagy-related 7; ATG9B, autophagy-related 9B; BAX, BCL2-associated X protein; CASP3, caspase 3, apoptosis-related cysteine peptidase; CAV1, caveolin 1, caveolae protein, 22kDa; CCL3, chemokine (C-C motif) ligand 3; CXCL1, chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity a); CXCR2, chemokine (C-X-C motif) receptor 2; DRAM2, DNA-damage regulated autophagy modulator 2; GFP-LC3B, green fluorescent protein-LC3B; IL12B, interleukin 12B; IL13, interleukin 13; IFNG, interferon, gamma; IPF, idiopathic pulmonary fibrosis; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 b; RELA, v-rel reticuloendotheliosis viral oncogene homolog A; SQSTM1, sequestosome 1; TGFB1, transforming growth factor, b 1; TGFBR2, transforming growth factor, b receptor II (70/80kDa); TNF, tumor necrosis factor; TUBB4, tubulin, b 4, class IV; WT, wild type.Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.

Background:In IPF MMP-1 is up-regulated and expressed in alveolar epithelial cells. Result: Transfection of MMP-1 in MLE cells increased proliferation/migration, protected from apoptosis, repressed oxygen consumption ratio and ROS production, and stimulated HIF-1␣. Conclusion: MMP-1 inhibits mitochondrial function and contributes to a proliferative/migratory and anti-apoptotic phenotype. Significance: MMP-1 promotes the Warburg effect characterized by increased aerobic glycolysis and HIF-1␣ during normoxia.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown etiology. A conspicuous feature is the formation and persistence of fibroblastic/myofibroblastic foci throughout the lung parenchyma. Mechanisms remain unknown, but data indicate that fibroblasts acquire an antiapoptotic phenotype. We hypothesized that transcriptional silencing of proapoptotic genes may be implicated, and accordingly we evaluated the epigenetic regulation of p14(ARF). The expression of p14(ARF) was analyzed by RT-PCR in IPF (n = 8) and normal derived fibroblasts (n = 4) before and after treatment with 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA). p14(ARF) gene promoter methylation was determined by methylation-specific PCR (MS-PCR) and by DNA digestion with endonuclease McrBc, which cleaves 50% of methylated CpG. Apoptosis was evaluated by Annexin-V and nuclear staining. p14(ARF) expression was significantly decreased in four of the eight IPF fibroblasts lines, which was restored after 5-aza treatment. No changes were found with TSA. MS-PCR of bisulfite-treated genomic DNA showed a correlation between the reduced expression of p14(ARF) and the presence of hypermethylated promoter. No amplification was observed in the DNA treated with the McrBc enzyme, corroborating promoter hypermethylation. p14(ARF)-hypermethylated IPF fibroblasts were significantly more resistant to staurosporine-and S-nitrosoglutathione-induced apoptosis compared with normal and nonmethylated IPF fibroblasts (P < 0.01) and showed reduced levels of p53. Resistance to apoptosis was provoked in fibroblasts when p14(ARF) expression was inhibited by siRNA (P < 0.05). These findings demonstrate that many IPF fibroblasts have reduced expression of the proapoptotic p14(ARF) attributable to promoter hypermethylation and indicate that epigenetic mechanisms may underlie their resistance to apoptosis.

Background Matrix metalloproteinases (MMPs) and C-reactive protein (CRP) are involved in chronic obstructive pulmonary disease (COPD) pathogenesis. The aim of the present work was to determine plasma concentrations of MMPs and CRP in COPD associated to biomass combustion exposure (BE) and tobacco smoking (TS). Methods Pulmonary function tests, plasma levels of MMP-1, MMP-7, MMP-9, MMP-9/TIMP-1 and CRP were measured in COPD associated to BE (n = 40) and TS (n =40) patients, and healthy non-smoking (NS) healthy women (controls, n = 40). Results Plasma levels of MMP-1, MMP-7, MMP-9, and MMP-9/TIMP-1 and CRP were higher in BE and TS than in the NS healthy women ( p <0.01 ). An inverse correlation between MMP-1, MMP-7, MMP-9, MMP-9/TIMP-1 and CRP plasma concentrations and FEV 1 was observed. Conclusions Increase of MMPs and CRP plasma concentrations in BE suggests a systemic inflammatory phenomenon similar to that observed in COPD associated to tobacco smoking, which may also play a role in COPD pathogenesis.