Meridew, Jeffrey A.

AbstractPulmonary fibrosis is characterized by pathological accumulation of scar tissue in the lung parenchyma. Many of the processes that are implicated in fibrosis, including increased extracellular matrix synthesis, also occur following pneumonectomy (PNX), but PNX instead results in regenerative compensatory growth of the lung. As fibroblasts are the major cell type responsible for extracellular matrix production, we hypothesized that comparing fibroblast responses to PNX and bleomycin (BLM) would unveil key differences in the role they play during regenerative versus fibrotic lung responses. RNA‐sequencing was performed on flow‐sorted fibroblasts freshly isolated from mouse lungs 14 days after BLM, PNX, or sham controls. RNA‐sequencing analysis revealed highly similar biological processes to be involved in fibroblast responses to both BLM and PNX, including TGF‐β1 and TNF‐α. Interestingly, we observed smaller changes in gene expression after PNX than BLM at Day 14, suggesting that the fibroblast response to PNX may be muted by expression of transcripts that moderate pro‐fibrotic pathways. Itpkc, encoding inositol triphosphate kinase C, was a gene uniquely up‐regulated by PNX and not BLM. ITPKC overexpression in lung fibroblasts antagonized the pro‐fibrotic effect of TGF‐β1. RNA‐sequencing analysis has identified considerable overlap in transcriptional changes between fibroblasts following PNX and those overexpressing ITPKC.

Abstract Idiopathic pulmonary fibrosis (IPF) is an aggressive and, thus far, incurable disease characterized by aberrant fibroblast-mediated extracellular matrix deposition. Our understanding of the disease etiology is incomplete; however, there is consensus that a reduction-oxidation (redox) imbalance plays a role. In this study, we use the autofluorescent properties of two redox molecules, NAD(P)H and FAD, to quantify changes in their relative abundance in living lung tissue of mice with experimental lung fibrosis and in freshly isolated cells from mouse lungs and humans with IPF. Our results identify cell population–specific intracellular redox changes in the lungs in experimental and human fibrosis. We focus particularly on redox changes within collagen-producing cells, where we identified a bimodal distribution of NAD(P)H concentrations, establishing NAD(P)Hhigh and NAD(P)Hlow subpopulations. NAD(P)Hhigh fibroblasts exhibited elevated profibrotic gene expression and decreased collagenolytic protease activity relative to NAD(P)Hlow fibroblasts. The NAD(P)Hhigh population was present in healthy lungs but expanded with time after bleomycin injury, suggesting a potential role in fibrosis progression. We identified a similar increased abundance of NAD(P)Hhigh cells in freshly dissociated lungs of subjects with IPF relative to control subjects, as well as similar reductions in collagenolytic activity in this cell population. These data highlight the complexity of redox state changes in experimental and human pulmonary fibrosis and the need for selective approaches to restore redox imbalances in the fibrotic lung.

AbstractPulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell‐specific approach. Our previous work demonstrated that targeting non‐canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non‐canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH‐associated PASMC remodeling, with longer‐lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non‐canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH.

Abstract Background Idiopathic pulmonary fibrosis (IPF) is characterized by pathological accumulation of scar tissue in the lung parenchyma. Many of the processes that are implicated in fibrosis, such as matrix deposition also occur following pneumonectomy (PNX). However, fibrosis does not occur. Since fibroblasts are the major cell type responsible for extracellular matrix production, we hypothesized that comparing their responses to PNX and bleomycin (BLM) would unveil key differences in the role they play during regulated and fibrotic lung growth. Methods RNA-sequencing was performed on flow cytometry-sorted fibroblasts from mouse lungs 14 days after bleomycin challenge, PNX, or sham. Pathway and transcription factor binding motif enrichment analysis were performed to characterize RNA-sequencing data. Normal human lung fibroblasts (NHLFs) were used as a cell culture model to validate targets. Results RNA-sequencing analysis revealed similar biological processes to be involved in both responses, including signaling by transforming growth factor-β (TGF-β1) and tumor necrosis factor-α. Transcription factor binding motif enrichment predicted erythroid transformation specific (ETS) superfamily members to play a key role in the response to BLM, whereas nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) were predicted to orchestrate much of the response to PNX. Itpkc, encoding inositol triphosphate kinase C, was a gene uniquely up-regulated by PNX and a likely AP-1 target. ITPKC overexpression in NHLFs antagonized the fibrotic effect of TGF-β1. RNA-sequencing analysis of primary NHLFs overexpressing ITPKC further supported its role in repressing collagen production and predicted it as an activator of canonical NF-κB signaling. ITPKC overexpression showed considerable overlap with the innate immune signaling seen following PNX. Conclusion Taken together, our RNA-sequencing analysis suggests that during post-PNX lung growth, AP-1 activates ITPKC to promote canonical NF-κB signaling to prevent fibrogenesis. Future studies to understand this lack of ITPKC induction during fibrotic injury may identify attractive therapeutic targets.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by myofibroblast accumulation and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-Seq on lung fibroblasts isolated from young and aged mice during the early resolution phase after bleomycin injury. We discovered that, relative to injured young fibroblasts, injured aged fibroblasts exhibited a profibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and its target nuclear factor of activated T cells-1 (NFATc1) as putative drivers of the sustained profibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts potentiated their fibrogenic activation, and this effect was attenuated by NFATc1 inhibition. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Interruption of PIM1 signaling in IPF lung explants ex vivo inhibited prosurvival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.

Rationale Idiopathic Pulmonary fibrosis (IPF) is characterized by progressive and ultimately fatal end‐stage lung scarring associated with uncontrolled deposition, stiffening and remodeling of fibrous connective tissue proteins. We have previously shown that agonism of the dopamine receptor D1 attenuates experimental lung fibrosis in mice, and that the lungs of IPF patients display reduced transcript and protein levels of DOPA decarboxylase (DDC), the enzyme that catalyzes the biosynthesis of dopamine. Here we investigate the role of endogenous dopamine production and signaling in the resolution of experimental lung fibrosis. Methods Using a self‐resolving model of experimental lung fibrosis, we administered bleomycin (1.1U/kg) to 10‐week old mice intratracheally at day 0, and collected lung tissue from sham and bleo groups at day 21 to establish the level of baseline fibrosis. We then administered the DDC inhibitor carbidopa (20mg/kg, IP), the D1 receptor antagonist SCH 39166 (0.5mg/kg, SC), or vehicle control daily from day 21 to 42. Lung tissue was collected for hydroxyproline and histological analysis. In order to investigate dopamine synthesis and signaling ex vivo, we generated precision‐cut lung slices (PCLS) from healthy mice and stimulated them +/‐ TGFβ for 72 hours and collected total RNA from the lung slices and conditioned media to measure dopamine production by ELISA. Results As previously observed, lung tissue collected at day 21 post injury displayed enhanced markers of fibrosis measured by hydroxyproline (478% increase over sham) and H&E stained histology. By day 42 the vehicle control mice resolved this fibrosis. In contrast, the mice treated with carbidopa or SCH 39166 maintained the fibrosis observed at day 21 (514% and 592% increase compared to sham, respectively). Consistent with our prior observation in IPF patient lung, PCLS cultured ex vivo with TGFβ expressed significantly less Ddc (75% reduction compared to control) and total dopamine abundance in the conditioned media dropped from ~120ng/mL to ~32ng/mL. Conclusions Dopamine synthesis through the activity of DDC, and agonism of the dopamine receptor D1 are essential to pulmonary fibrosis resolution after injury. PCLS represents a useful model to further investigate the molecular mechanisms leading to reduced expression of DDC, and test pharmacological approaches to stimulate dopamine signaling as a potential therapy for IPF.