Abstract
Objective: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. The microenvironment (e.g. ECM) plays a significant role in IPF progression. We developed a novel in-vitro biological system of IPF fibroblast conditioned matrix (CM), which enables the study of ECM changes on fibroblast cell phenotype and signaling.
Methods: IPF tissue-derived primary fibroblasts were cultured on Matrigel and then cleared by NH4OH, thus creating the IPF-CM. Normal tissue primary fibroblast CM served as control. Normal fibroblasts were cultured on these matrixes for 3, 24 & 72 hrs. We tested: cell count and viability (trypan blue, FACS), cell distribution (visual assessment, ImageJ), TGFβ pathway activation (pSmad3), αSMA and Collagen1a expression (Western blot).
Results: IPF-CM induced large aggregate formation (243% increase, p<0.05, 24 hrs). This was a result of elevated cell number (53%, p<0.05), increased viability (15%, p<0.05) and cell migration (microscopic observations at earlier time points). In addition, we found that the IPF-CM increased pSmad3 levels (280%, p<0.05) following 3 & 24 hrs. While αSMA levels were not changed, Collagen1a expression was increased in cells cultured on the IPF-CM in comparison to control (340%, p<0.05, 24 hrs).
Conclusions: IPF fibroblasts alter the ECM differently than normal fibroblasts, thus creating a CM that further propagates 'IPF like' phenotype in normal fibroblasts. Better understanding of these processes can lead to the design of anti-fibrotic treatments.
- Copyright ©the authors 2016