A model of human lung fibrogenesis for the assessment of anti-fibrotic strategies in idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease with limited therapeutic options. KCa3.1 ion channels play a critical role in TGFβ1-dependent pro-fibrotic responses in human lung myofibroblasts. We aimed to develop a human lung parenchymal model of fibrogenesis and test the efficacy of the selective KCa3.1 blocker senicapoc. 2 mm3 pieces of human lung parenchyma were cultured for 7 days in DMEM ± TGFβ1 (10 ng/ml) and pro-fibrotic pathways examined by RT-PCR, immunohistochemistry and collagen secretion. Following 7 days of culture with TGFβ1, 41 IPF- and fibrosis-associated genes were significantly upregulated. Immunohistochemical staining demonstrated increased expression of ECM proteins and fibroblast-specific protein after TGFβ1-stimulation. Collagen secretion was significantly increased following TGFβ1-stimulation. These pro-fibrotic responses were attenuated by senicapoc, but not by dexamethasone. This 7 day ex vivo model of human lung fibrogenesis recapitulates pro-fibrotic events evident in IPF and is sensitive to KCa3.1 channel inhibition. By maintaining the complex cell-cell and cell-matrix interactions of human tissue, and removing cross-species heterogeneity, this model may better predict drug efficacy in clinical trials and accelerate drug development in IPF. KCa3.1 channels are a promising target for the treatment of IPF.

Ca2+ signalling in fibroblasts and the therapeutic potential of KCa3.1 channel blockers in fibrotic diseases

“…In a human model of lung fibrosis, 44 IPF‐ and fibrosis‐associated genes were significantly up‐regulated by TGF‐β1 at the mRNA level, supported by immunohistochemical staining of the tissue for extracellular matrix proteins, α‐ smooth muscle actin and fibroblast‐specific protein (Roach et al, ). Collagen secretion was significantly increased following TGF‐β1 stimulation.…”

“…Collagen secretion was significantly increased following TGF‐β1 stimulation. These pro‐fibrotic responses were markedly attenuated by the selective K Ca 3.1 blocker senicapoc but not by dexamethasone (Roach et al, ; Figure ). In support of this, in a sheep model of bleomycin‐induced pulmonary fibrosis in separate lung segments, where each sheep acts as its own control, oral administration of senicapoc for 2 weeks after bleomycin instillation improved pre‐established bleomycin‐induced fibrosis with associated improvements in extracellular matrix and collagen deposition, α‐ smooth muscle actin expression and lung function (Organ et al, ).…”

“…Heatmaps displaying the effects of TGF‐β1 on fibrosis‐associated gene expression ( n = 23 donors) and the inhibitory effects of senicapoc (100 nM; n = 11 donors) in a human model of lung fibrosis. Twenty‐eight genes up‐regulated by TGF‐β1 were significantly down‐regulated following treatment with senicapoc for 7 days in comparison with TGF‐β1‐stimulation alone (adapted from Roach et al, )…”

“…K Ca 3.1 channel mRNA and the number of functional channels are F I G U R E 7 Heatmaps displaying the effects of TGF-β1 on fibrosis-associated gene expression (n = 23 donors) and the inhibitory effects of senicapoc (100 nM; n = 11 donors) in a human model of lung fibrosis. Twenty-eight genes up-regulated by TGF-β1 were significantly downregulated following treatment with senicapoc for 7 days in comparison with TGF-β1-stimulation alone (adapted from Roach et al, 2018) increased in human airway smooth muscle cells following activation with TGF-β1 (Shepherd et al, 2007). Pharmacological inhibition of K Ca 3.1 in these cells inhibited growth factor-dependent proliferation by arresting cell cycle progression, which normally requires membrane hyperpolarisation and increased Ca 2+ influx during G 1 to allow the cells to pass through the G 1 -S checkpoint (Shepherd et al, 2007;Wonderlin & Srobl, 1996) Senicapoc-treated sheep also demonstrated reduced non-specific airway hyperresponsiveness to carbachol following allergen challenge.…”

“…Both non-fibrotic and IPFderived lung myofibroblasts express αsmooth muscle actin at "rest,"and in the presence of K Ca 3.1 blockers, this expression can be reduced, returning myofibroblasts towards a quiescent fibroblast phenotype(Roach et al, 2013; Figure 6). These effects were attributed to the inhibition of Smad2/3 signalling pathways by K Ca 3.1 blockade, with reduced constitutive and TGF-β1-dependent Smad2/3 phosphorylation and nuclear translocation, as well reduced Smad2/3dependent transcription of pro-fibrotic genes, such as αsmooth muscle actin and collagen type-1, processes that were also dependent on extracellular Ca 2+(Roach, Feghali-Bostwick, Wulff, Amrani, & Braddin, 2015).In a human model of lung fibrosis, 44 IPF-and fibrosis-associated genes were significantly up-regulated by TGF-β1 at the mRNA level, supported by immunohistochemical staining of the tissue for extracellular matrix proteins, αsmooth muscle actin and fibroblast-specific protein(Roach et al, 2018). Collagen secretion was significantly increased following TGF-β1 stimulation.…”