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Extracellular matrix remodeling associated with bleomycin-induced lung injury supports pericyte-to-myofibroblast transition.

ABSTRACT: Of the many origins of pulmonary myofibroblasts, microvascular pericytes are a known source. Prior literature has established the ability of pericytes to transition into myofibroblasts, but provide limited insight into molecular cues that drive this process during lung injury repair and fibrosis. Fibronectin and RGD-binding integrins have long been considered pro-fibrotic factors in myofibroblast biology, and here we test the hypothesis that these known myofibroblast cues coordinate pericyte-to-myofibroblast transitions. Specifically, we hypothesized that αvβ3 integrin engagement on fibronectin induces pericyte transition into myofibroblastic phenotypes in the murine bleomycin lung injury model. Myosin Heavy Chain 11 (Myh11)-CreERT2 lineage tracing in transgenic mice allows identification of cells of pericyte origin and provides a robust tool for isolating pericytes from tissues for further evaluation. We used this murine model to track and characterize pericyte behaviors during tissue repair. The majority of Myh11 lineage-positive cells are positive for the pericyte surface markers, PDGFRβ (55%) and CD146 (69%), and display typical pericyte morphology with spatial apposition to microvascular networks. After intratracheal bleomycin treatment of mice, Myh11 lineage-positive cells showed significantly increased contractile and secretory markers, as well as αv integrin expression. According to RNASeq measurements, many disease and tissue-remodeling genesets were upregulated in Myh11 lineage-positive cells in response to bleomycin-induced lung injury. In vitro, blocking αvβ3 binding through cycloRGDfK prevented expression of the myofibroblastic marker αSMA relative to controls. In response to RGD-containing provisional matrix proteins present in lung injury, pericytes may alter their integrin profile.

SUBMITTER: Hannan RT

PROVIDER: S-EPMC8233458 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and aimsPerianal fistula represents one of the most disabling manifestations of Crohn's disease (CD) due to complete destruction of the affected mucosa, which is replaced by granulation tissue and associated with changes in tissue organization. To date, the molecular mechanisms underlying perianal fistula formation are not well defined. Here, we dissected the tissue changes in the fistula area and addressed whether a dysregulation of extracellular matrix (ECM) homeostasis can support fistula formation.MethodsSurgical specimens from perianal fistula tissue and the surrounding region of fistulizing CD were analyzed histologically and by RNA sequencing. Genes significantly modulated were validated by real-time polymerase chain reaction, Western blot, and immunofluorescence assays. The effect of the protein product of TNF-stimulated gene-6 (TSG-6) on cell morphology, phenotype, and ECM organization was investigated with endogenous lentivirus-induced overexpression of TSG-6 in Caco-2 cells and with exogenous addition of recombinant human TSG-6 protein to primary fibroblasts from region surrounding fistula. Proliferative and migratory assays were performed.ResultsA markedly different organization of ECM was found across fistula and surrounding fistula regions with an increased expression of integrins and matrix metalloproteinases and hyaluronan (HA) staining in the fistula, associated with increased newly synthesized collagen fibers and mechanosensitive proteins. Among dysregulated genes associated with ECM, TNFAI6 (gene encoding for TSG-6) was as significantly upregulated in the fistula compared with area surrounding fistula, where it promoted the pathological formation of complexes between heavy chains from inter-alpha-inhibitor and HA responsible for the formation of a crosslinked ECM. There was a positive correlation between TNFAI6 expression and expression of mechanosensitive genes in fistula tissue. The overexpression of TSG-6 in Caco-2 cells promoted migration, epithelial-mesenchymal transition, transcription factor SNAI1, and HA synthase (HAs) levels, while in fibroblasts, isolated from the area surrounding the fistula, it promoted an activated phenotype. Moreover, the enrichment of an HA scaffold with recombinant human TSG-6 protein promoted collagen release and increase of SNAI1, ITGA4, ITGA42B, and PTK2B genes, the latter being involved in the transduction of responses to mechanical stimuli.ConclusionsBy mediating changes in the ECM organization, TSG-6 triggers the epithelial-mesenchymal transition transcription factor SNAI1 through the activation of mechanosensitive proteins. These data point to regulators of ECM as new potential targets for the treatment of CD perianal fistula.

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Extracellular matrix remodeling associated with bleomycin-induced lung injury supports pericyte-to-myofibroblast transition.

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