Enhanced asthma-related fibroblast to myofibroblast transition is the result of profibrotic TGF-β/Smad2/3 pathway intensification and antifibrotic TGF-β/Smad1/5/(8)9 pathway impairment

Abstract

Airway remodelling with subepithelial fibrosis, which abolishes the physiological functions of the bronchial wall, is a major issue in bronchial asthma. Human bronchial fibroblasts (HBFs) derived from patients diagnosed with asthma display in vitro predestination towards TGF-β1-induced fibroblast-to-myofibroblast transition (FMT), a key event in subepithelial fibrosis. As commonly used anti-asthmatic drugs do not reverse the structural changes of the airways, and the molecular mechanism of enhanced asthma-related TGF-β1-induced FMT is poorly understood, we investigated the balance between the profibrotic TGF-β/Smad2/3 and the antifibrotic TGF-β/Smad1/5/9 signalling pathways and its role in the myofibroblast formation of HBF populations derived from asthmatic and non-asthmatic donors. Our findings showed for the first time that TGF-β-induced activation of the profibrotic Smad2/3 signalling pathway was enhanced, but the activation of the antifibrotic Smad1/5/(8)9 pathway by TGF-β1 was significantly diminished in fibroblasts from asthmatic donors compared to those from their healthy counterparts. The impairment of the antifibrotic TGF-β/Smad1/5/(8)9 pathway in HBFs derived from asthmatic donors was correlated with enhanced FMT. Furthermore, we showed that Smad1 silencing in HBFs from non-asthmatic donors increased the FMT potential in these cells. Additionally, we demonstrated that activation of antifibrotic Smad signalling via BMP7 or isoliquiritigenin [a small-molecule activator of the TGF-β/Smad1/5/(8)9 pathway] administration prevents FMT in HBFs from asthmatic donors through downregulation of profibrotic genes, e.g., α-SMA and fibronectin. Our data suggest that influencing the balance between the antifibrotic and profibrotic TGF-β/Smad signalling pathways using BMP7-mimetic compounds presents an unprecedented opportunity to inhibit subepithelial fibrosis during airway remodelling in asthma.

Asthma-derived fibroblast to myofibroblast transition is enhanced in comparison to fibroblasts derived from non-asthmatic patients in 3D in vitro culture due to Smad2/3 signalling

“…We also showed, for the first time, a profound difference in α-SMA mRNA expression between TGF-β-treated HBFs from AS and NA group cultured in 3D collagen gel, even though the changes of their morphology in comparison to unstimulated cells were not observed. However, this effect was less pronounced in 3D collagen gel in comparison to our earlier results with the 2D model (Michalik et al, 2009, Paw et al 2017, Wnuk et al, 2020. It was previously demonstrated that the compliance of collagen gels regulated TGF-β-induction of α-SMA expression in human gingival fibroblasts (Arora et al, 1999).…”

“…Moreover, fibroblasts from asthmatics, as in the '2D' model, produce more extracellular matrix proteins (COL1, FN1) when compared to their non-asthmatic counterparts. Similarly to the '2D' model (Paw et al, 2017(Paw et al, , 2018Wnuk et al, 2020), this is due to the increased activation of the TGF-β/Smad2/3 pathway, which is a canonical pathway through which FMT induction occurs. All of the processes described above are less pronounced than in the '2D' model, which may be related to the insufficiently rigid substratum, but the differences between cells from asthmatics and non-asthmatics are evident.…”

“…ling pathway was crucial for FMT (Michalik et al, 2013;Wójcik-Pszczoła et al, 2016;Wnuk et al, 2020). Since we observed an enhanced TGF-β 1 -induced expression of profibrotic genes in HBFs derived from asthmatic patients when compared to non-asthmatic donors, we further focused on analysis of nuclear localisation of p-Smad2/3 between HBFs derived from both studied groups, in the developed 3D model.…”

Responsiveness of human bronchial fibroblasts and epithelial cells from asthmatic and non-asthmatic donors to the transforming growth factor-β1 in epithelial-mesenchymal trophic unit model

“…Our longitudinal studies clearly show that populations of the HBF from AS and NA cultured in identical conditions in a serum-free medium display different features affecting their sensitivity to the external TGF-β 1 and FMT potential (20,28,30). In standard in vitro cultures the HBFs AS in response to the TGF-β 1 develop the myo broblastic phenotype characterized by a relatively high level of α-SMA incorporated into stress bers more e ciently than the NA counterparts (23)(24)(25)27,28).…”

“…Longitudinal studies on the TGF-β 1 -induced FMT potential (a key event in asthmatic subepithelial brosis) revealed a facilitated ability of the asthmatic HBF populations in standard '2D' cultures for phenotypic shifts (23,25,27,28,30,49), that may indicate the involvement of intrinsic properties of these cells in the regulation of FMT. In this study, we also observed the enhanced FMT potential in the TGF-β 1simulated HBF AS populations in contrast to the NA counterparts, when cells were grown in a monoculture as well as in compatible EMTUs.…”