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  • PSI-6206 receptor Next we asked how a common activation mark

    2021-10-15

    Next, we asked how a common activation marker of fibroblasts, Acta2, defined gene expression signatures in alveolar airspaces by using Acta2-mKO1 reporter mice (Fig. 2A). In the lungs of Acta2-mKO1 mice, mKO1 was expressed in smooth muscle cells and mesenchymal cells in PSI-6206 receptor (Fig. 2B). By sorting mKO1 low, intermediate, and high population, we confirmed that mKO1 expression correlated with Acta2 mRNA expression (Supplementary Fig. 1). We crossed Col-GFP mice with Acta2-mKO1 mice, and transferred lung fibroblasts isolated from those mice into BLM-treated wild type mice. We sorted mKO1 high and low population from the host lungs 4 days after the transfer, and performed transcriptome analysis (Fig. 2C). We calculated fold gene expression changes between mKO1 high and low populations and examined upstream regulators for genes upregulated (>2 folds) in the mKO1 high population (Table 2). Similar to the later time points of the previous experiment, Klf4 and Gli signaling molecules were enriched as upstream regulators. GO term analysis also showed higher expression of genes related to ECM organization in mKO1 high population (Supplementary Table 4). These data suggest that Acta2 high activated fibroblasts highly produce ECM and those activation genes are possibly regulated by Klf4 or Gli signaling molecules. To test how upregulated genes after exposure to alveolar airspaces affect scar formation in fibrosis, we utilized GANT61, an inhibitor of Gli1 and Gli2, in the BLM-induced lung fibrosis model. GANT61 was previously shown to partially reduce ECM deposition in BLM-induced lung fibrosis [17] and unilateral ureteral obstruction kidney fibrosis [9]. We treated Col-GFP mice with BLM and injected GANT61 every other day from day 5–13 after BLM treatment (Fig. 3A). We found that GANT61 treatment altered the pattern of scars on day 14 (Fig. 3B and C). The lungs treated with GANT61 were characterized by dilated airspaces with partially thickened interstitial regions, which is in stark contrast to the dense scars with less airspaces in vehicle-treated mice. Thickened interstitial regions in GANT61-treated mice encompassed collagen 1 matrix but with smaller Col-GFP+ clusters compared to vehicle-treated mice (Fig. 3C). Increased airspaces in GANT61-treated mice compared to vehicle was also illustrated by image quantification (Fig. 3D). These data may suggest that inhibition of Gli signalings affect fibroblast activation and alter scar formation. To explore how GANT61 affects fibroblast activation, we treated Col-GFP mice with GANT61 on day 7 after BLM treatment, and measured migration capacity of Col-GFP+ cells purified from the lungs on day 9, which is the phase that alveolar fibroblasts migrate into alveolar airspaces (Fig. 4A) [6]. As expected, Col-GFP+ cells from GANT61-treated mice showed reduced migration capacity compared to those from vehicle-treated mice (Fig. 4B and C). These data suggest that one of the mechanisms of altered scar formation by Gli signaling inhibition is the reduction of migration capacity in activated fibroblasts in alveolar airspaces.
    Discussion Our previous study showed that intratracheal transfer of lung fibroblasts in bleomycin-induced lung fibrosis is useful in modeling the activation of fibroblasts [6]. Since the migration of interstitial fibroblasts into alveolar airspaces is an important process in forming fibrotic niche at injured sites [5], we utilized intratracheal transfer to explore molecular changes of fibroblasts in alveolar airspaces. We found that Hif1a binding motifs were enriched in promoter regions of genes upregulated in the initial phase after the transfer. Hif1a is essential in responding to hypoxia, and its various roles are reported in multiple cell types in fibrosis [18]. Fibroblasts undergo drastic morphological changes after exposure to injured alveolar airspaces, characterized by enlarged cell size and rounded shape [6]. Since Hif1a activation in fibroblasts enlarges cell size and rearranges actin filaments [19], it is possible that metabolic and translational changes caused by Hif1a lead to the morphological change in the initial activation phase. An open question is how Hif1a is activated in injured alveolar airspaces. Although the canonical hypoxia pathway may occur, the degree of hypoxia in injured alveolar airspaces is unknown. Another possibility is that iron sequestration mediated by ferritin activates Hif1a [20], as altered iron metabolism is associated with lung diseases [21]. These questions should be addressed.