Novel combination of collagen dynamics analysis and transcriptional profiling reveals fibrosis-relevant genes and pathways

Marjolein E. Blaauboer, Claire L. Emson, Lars Verschuren, Marjan van Erk, Scott M. Turner, Vincent Everts, Roeland Hanemaaijer, Reinout Stoop

Collagen deposition is a key process during idiopathic pulmonary fibrosis; however, little is known about the dynamics of collagen formation during disease development. Tissue samples of early stages of human disease are not readily available and it is difficult to identify changes in collagen content, since standard collagen analyses do not distinguish between 'old' and 'new' collagen. Therefore, the current study aimed to (i) investigate the dynamics of new collagen formation in mice using bleomycin-induced lung fibrosis in which newly synthesized collagen was labeled with deuterated water and (ii) use this information to identify genes and processes correlated to new collagen formation. Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at 1 to 5 weeks after fibrosis induction. Collagen synthesized during the week before sacrifice was labeled with deuterium by providing mice with deuterated drinking water. After sacrifice, we collected lung tissue for microarray analysis, determination of new collagen formation, and histology. Furthermore, we measured in vitro the expression of selected genes after transforming growth factor (TGF) β₁-induced myofibroblast differentiation. Deuterated water labeling showed a strong increase in new collagen formation already during the first week after fibrosis induction and a complete return to baseline at five weeks. Correlation of new collagen formation data with gene expression data allowed us to create a gene expression signature of fibrosis within the lung and revealed fibrosis-specific processes, among which proliferation. This was confirmed by measuring cell proliferation and collagen synthesis simultaneously using deuterated water incorporation in a separate experiment. Furthermore, new collagen formation strongly correlated with gene expression of e.g. elastin, Wnt-1 inducible signaling pathway protein 1, tenascin C, lysyl oxidase, and type V collagen. Gene expression of these genes was upregulated in vitro in fibroblasts stimulated with TGFβ₁. Together, these data demonstrate, using a novel combination of technologies, that the core process of fibrosis, i.e. the formation of new collagen, correlates not only with a wide range of genes involved in general extracellular matrix production and modification but also with cell proliferation. The observation that the large majority of the genes which correlated with new collagen formation also were upregulated during TGFβ₁-induced myofibroblast differentiation provides further evidence for their involvement in fibrosis.