Vanesa Vicens-Zygmunt

Lung fibrotic tenascin-C upregulation is associated with other extracellular matrix proteins and induced by TGFβ1

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive parenchymal lung disease of unknown aetiology and poor prognosis, characterized by altered tissue repair and fibrosis. The extracellular matrix (ECM) is a critical component in regulating cellular homeostasis and appropriate wound healing. The aim of our study was to determine the expression profile of highlighted ECM proteins in IPF lungs.MethodsECM gene and protein expression was analyzed by cDNA microarrays, rt-PCR, immunohistochemistry and western-blot in lungs from idiopathic pulmonary fibrosis (IPF), hypersensitivity pneumonitis (HP), categorized as chronic (cHP) and subacute (saHP), and healthy lung tissue. Primary fibroblast cultures from normal subjects and fibrotic patients were studied to evaluate tenascin-C (TNC) synthesis.ResultsA total of 20 ECM proteins were upregulated and 6 proteins downregulated in IPF. TNC was almost undetected in normal lungs and significantly upregulated in fibrotic lungs (IPF and cHP) compared to saHP. Furthermore, it was located specifically in the fibroblastic foci areas of the fibrotic lung with a subepithelial gradient pattern. TNC levels were correlated with fibroblastic foci content in cHP lungs. Versican and fibronectin glycoproteins were associated with TNC, mainly in fibroblastic foci of fibrotic lungs. Fibroblasts from IPF patients constitutively synthesized higher levels of TNC than normal fibroblasts. TNC and α-sma was induced by TGF-β1 in both fibrotic and normal fibroblasts. TNC treatment of normal and fibrotic fibroblasts induced a non-significant increased α-sma mRNA.ConclusionsThe difference in ECM glycoprotein content in interstitial lung diseases could contribute to the development of lung fibrosis. The increase of TNC in interstitial areas of fibrotic activity could play a key role in the altered wound healing.

Increased AGE-RAGE ratio in idiopathic pulmonary fibrosis

BackgroundThe abnormal epithelial-mesenchymal restorative capacity in idiopathic pulmonary fibrosis (IPF) has been recently associated with an accelerated aging process as a key point for the altered wound healing. The advanced glycation end-products (AGEs) are the consequence of non-enzymatic reactions between lipid and protein with several oxidants in the aging process. The receptor for AGEs (RAGEs) has been implicated in the lung fibrotic process and the alveolar homeostasis. However, this AGE-RAGE aging pathway has been under-explored in IPF.MethodsLung samples from 16 IPF and 9 control patients were obtained through surgical lung biopsy. Differences in AGEs and RAGE expression between both groups were evaluated by RT-PCR, Western blot and immunohistochemistry. The effect of AGEs on cell viability of primary lung fibrotic fibroblasts and alveolar epithelial cells was assessed. Cell transformation of fibrotic fibroblasts cultured into glycated matrices was evaluated in different experimental conditions.ResultsOur study demonstrates an increase of AGEs together with a decrease of RAGEs in IPF lungs, compared with control samples. Two specific AGEs involved in aging, pentosidine and Nε-Carboxymethyl lysine, were significantly increased in IPF samples. The immunohistochemistry identified higher staining of AGEs related to extracellular matrix (ECM) proteins and the apical surface of the alveolar epithelial cells (AECs) surrounding fibroblast foci in fibrotic lungs. On the other hand, RAGE location was present at the cell membrane of AECs in control lungs, while it was almost missing in pulmonary fibrotic tissue. In addition, in vitro cultures showed that the effect of AGEs on cell viability was different for AECs and fibrotic fibroblasts. AGEs decreased cell viability in AECs, even at low concentration, while fibroblast viability was less affected. Furthermore, fibroblast to myofibroblast transformation could be enhanced by ECM glycation.ConclusionsAll of these findings suggest a possible role of the increased ratio AGEs-RAGEs in IPF, which could be a relevant accelerating aging tissue reaction in the abnormal wound healing of the lung fibrotic process.

Identification of MMP28 as a biomarker for the differential diagnosis of idiopathic pulmonary fibrosis

Background and objective Idiopathic Pulmonary Fibrosis (IPF) is a progressive disease of unknown etiology. The diagnosis is based on the identification of a pattern of usual interstitial pneumonia either by high resolution computed tomography and/or histology. However, a similar pattern can be observed in other fibrotic lung disorders, and precise diagnosis remains challenging. Studies on biomarkers contributing to the differential diagnosis are scanty, and still in an exploratory phase. Our aim was to evaluate matrix metalloproteinase (MMP)-28, which has been implicated in abnormal wound healing, as a biomarker for distinguishing IPF from fibrotic non-IPF patients. Methods The cell localization of MMP28 in lungs was examined by immunohistochemistry and its serum concentration was measured by ELISA in two different populations. The derivation cohort included 82 IPF and 69 fibrotic non-IPF patients. The validation cohort involved 42 IPF and 41 fibrotic non-IPF patients. Results MMP28 was detected mainly in IPF lungs and localized in epithelial cells. In both cohorts, serum concentrations of MMP28 were significantly higher in IPF versus non-IPF (mostly with lung fibrosis associated to autoimmune diseases and chronic hypersensitivity pneumonitis) and healthy controls (ANOVA, p<0.0001). The AUC of the derivation cohort was 0.718 (95%CI, 0.635–0.800). With a cutoff point of 4.5 ng/mL, OR was 5.32 (95%CI, 2.55–11.46), and sensitivity and specificity of 70.9% and 69% respectively. The AUC of the validation cohort was 0.690 (95%CI, 0.581–0.798), OR 4.57 (95%CI, 1.76–12.04), and sensitivity and specificity of 69.6% and 66.7%. Interestingly, we found that IPF patients with definite UIP pattern on HRCT showed higher serum concentrations of MMP28 than non-IPF patients with the same pattern (7.8±4.4 versus 4.9±4.4; p = 0.04). By contrast, no differences were observed when IPF with possible UIP-pattern were compared (4.7±3.2 versus 3.9±3.0; p = 0.43). Conclusion These findings indicate that MMP28 might be a useful biomarker to improve the diagnostic certainty of IPF.

Anti-fibrotic effects of pirfenidone and rapamycin in primary IPF fibroblasts and human alveolar epithelial cells

BackgroundPirfenidone, a pleiotropic anti-fibrotic treatment, has been shown to slow down disease progression of idiopathic pulmonary fibrosis (IPF), a fatal and devastating lung disease. Rapamycin, an inhibitor of fibroblast proliferation could be a potential anti-fibrotic drug to improve the effects of pirfenidone.MethodsPrimary lung fibroblasts from IPF patients and human alveolar epithelial cells (A549) were treated in vitro with pirfenidone and rapamycin in the presence or absence of transforming growth factor β1 (TGF−β). Extracellular matrix protein and gene expression of markers involved in lung fibrosis (tenascin-c, fibronectin, collagen I [COL1A1], collagen III [COL3A1] and α-smooth muscle actin [α-SMA]) were analyzed. A cell migration assay in pirfenidone, rapamycin and TGF−β-containing media was performed.ResultsGene and protein expression of tenascin-c and fibronectin of fibrotic fibroblasts were reduced by pirfenidone or rapamycin treatment. Pirfenidone-rapamycin treatment did not revert the epithelial to mesenchymal transition pathway activated by TGF−β. However, the drug combination significantly abrogated fibroblast to myofibroblast transition. The inhibitory effect of pirfenidone on fibroblast migration in the scratch-wound assay was potentiated by rapamycin combination.ConclusionsThese findings indicate that the combination of pirfenidone and rapamycin widen the inhibition range of fibrogenic markers and prevents fibroblast migration. These results would open a new line of research for an anti-fibrotic combination therapeutic approach.