Defining molecular signatures of fibroblast phenotypes in fibrotic lung disease

Introduction: Fibroblasts exhibit an extraordinary capacity to undergo phenotypic changes during development and disease, both in in vitro and in vivo settings thereof. These changes include altered motility, migration, activation, or apoptosis. Enhanced migratory capacity e.g. of primary lung fibroblasts in Idiopathic Pulmonary Fibrosis (IPF) patients was found in vitro, but the underlying mechanisms thereof remain elusive. Identification of aberrant molecular signaling pathways in migrating fibroblasts of fibrotic lungs may contribute to effective novel treatments for IPF.

Methods: We have established a 3D cell culture invasion model, which enables the complete separation of invading from non-invading fibroblasts. Membrane inserts with a pore diameter of 8µm are coated with collagen matrices (0.8mg/ml) on the bottom of the membrane. CCL206 lung fibroblasts are plated on top of the membrane inserts opposite to the collagen matrix in DMEM/-F12 with 1% FCS. The cells are then left to invade the collagen for 72h. Finally, separation of membranes and collagen matrices allows the distinguished analysis of RNA/protein expression levels of invading fibroblasts (matrix) and non-invading fibroblasts (membrane).

Results: We observed that the invading and non-invading fibroblasts are morphologically distinguishable. Furthermore, we showed that the invasion capacity of fibroblasts is augmented by the IPF related growth factors TGFβ₁ (transforming growth factor β₁) and EGF (epidermal growth factor) treatment. Gene expression analysis (by qRT-PCR) of the separated fractions demonstrated different expression profiles. Notably, CXCR4, a chemokine receptor important for cell migration in IPF was found to be significantly upregulated in the invading fibroblast fraction.

Discussion/Outlook: Lung fibroblasts show two morphologically distinguishable subpopulations in an in vitro 3D environment and an increased invasion capacity after TGFβ₁ and EGF treatment. Furthermore, the two subpopulations show variations in gene expression. To broaden the understanding of the gene expression profile of invading fibroblasts, microarray analysis will be employed. Knock-down of genes, found to be upregulated in the invading subpopulation will lead to a better understanding of the underlying mechanism.