Transcriptome analysis of IPF fibroblastic foci identifies key pathways involved in fibrogenesis

Abstract

IntroductionFibroblastic foci represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling unmanipulated myofibroblasts within fibrotic foci in situ by laser capture microdissection.MethodsThe challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene-based approach to identify transcriptional signatures, which correlate with fibrillar collagen gene expression.ResultsWGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that transforming growth factor β1 (TGF-β1), RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene-edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-β1-induced mechanistic target of rapamycin complex 1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts.ConclusionThese data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identify the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions.

Proteome Characterization of BALF Extracellular Vesicles in Idiopathic Pulmonary Fibrosis: Unveiling Undercover Molecular Pathways

“…Our identification of several Rho GTPases in EVs suggests their potential action on cytoskeleton remodeling by mediating actin filament rearrangement via ROCK [52]. Indeed, interesting studies report that ROCK signaling pathways are involved in myofibroblast differentiation and fibrogenic processes, especially pulmonary fibrosis such as IPF [40,53,54]. Curiously, given the growing attention to Wnt signaling in the regulation of cellular adhesions and its involvement in IPF pathogenic mechanisms, Franco et al reported that Rho GTPases' regulation modulates cell migration and polarity via a βcatenin-independent Wnt pathway [55].…”

The IPF fibroblastic focus is an active collagen biosynthesis factory embedded in a distinct extracellular matrix

“…A previous study using laser capture microdissection of the fibroblastic focus coupled with RNA-sequencing and a novel weighted gene co-expression network analysis showed elevation of genes associated with cell cycle, inflammation/differentiation, translation, and cytoskeleton/cell adhesion [54]. Our analysis identified 100 proteins increased and 211 decreased in the IPF FF relative to non-fibrotic alveoli control (Figure 5A; a full list in Supplemental File 2).…”

Proteome Characterization of BALF Extracellular Vesicles in Idiopathic Pulmonary Fibrosis: Unveiling Undercover Molecular Pathways

“…Our identification of several Rho GTPases in EVs suggests their potential action on cytoskeleton remodeling by mediating actin filament rearrangement via ROCK [52]. Indeed, interesting studies report that ROCK signaling pathways are involved in myofibroblast differentiation and fibrogenic processes, especially pulmonary fibrosis such as IPF [40,53,54]. Curiously, given the growing attention to Wnt signaling in the regulation of cellular adhesions and its involvement in IPF pathogenic mechanisms, Franco et al reported that Rho GTPases' regulation modulates cell migration and polarity via a β-catenin-independent Wnt pathway [55].…”