Document Type
Article
Publication Date
11-22-2025
Abstract
Background
Injury-repair responses typically induce tissue or organ scarring. Generally, cAMP-mobilizing agents inhibit the activation of fibroblasts and deposition of extracellular matrix. Some but not all cAMP-mobilizing agents inhibit fibrosis. Evidence suggests that inhaled treprostinil, a prostacyclin (IP) analog, increases intracellular cAMP levels [cAMP]I, and slows the decline in pulmonary function in patients with idiopathic pulmonary fibrosis (IPF). However, the molecular mechanisms by which cAMP-mobilizing agents, including treprostinil, alter the expression of matrix proteins in human lung fibroblasts (HLF) remain unclear. Unlike other Gαs -coupled receptors, we posit that the antifibrotic properties of treprostinil are driven by cAMP-mobilizing-dependent and -independent responses mediated by the IP receptor activation.
Methods
As a model of lung fibrosis, primary HLF derived from non-IPF and IPF donors were stimulated with TGF-β; collagen 1A1 and plasminogen activator inhibitor-1 (PAI) expression were then measured in the presence and absence of cAMP mobilizing agents. The necessity of receptor activation for inhibiting TGF-β-induced markers of fibrosis was determined by using soluble receptor inhibitors and decreasing receptor expression with siRNA.
Results
Treprostinil decreased TGF-β-induced extracellular matrix production by HLF, and the magnitude of the inhibition was greater than that of other cAMP-mobilizing GPCR agonists despite these agents comparably increasing cAMP levels. There was no difference in the sensitivity and magnitude of the treprostinil inhibition in HLF derived from non-fibrosis and lung fibrosis donors. Treprostinil inhibition of TGF-β-induced collagen 1A1and PAI-1 was mediated through the activation of the IP receptor. The activation of the EP2 receptor, in part, inhibited TGF-β-induced collagen 1A1 expression by treprostinil or prostaglandin E2. β2 agonists had little effect on TGF-β-induced expression of collagen 1A1 and PAI-1. The inhibitory effects of treprostinil on TGF-β-induced collagen 1A1 expression required Gαs activation, while Gαs only partially mediated treprostinil inhibition of PAI-1.
Conclusion
The anti-fibrotic properties of treprostinil are primarily mediated by the IP receptor, acting through both Gαs-dependent and -independent pathways. Understanding the differential effects of cAMP-mobilizing pathways on HLF fibrotic signatures can provide insight into developing novel targets to manage IPF.
Recommended Citation
Orfanos, S., Deeney, B.T., Cao, G. et al. The differential effects of cAMP mobilizing agents on TGF-β-induced extracellular matrix in human lung-derived fibroblasts: insights into therapeutic targets for lung fibrosis. Respir Res 26, 328 (2025). https://doi.org/10.1186/s12931-025-03387-3
Supplementary Material 1: Supplemental figure 1. Immunohistochemistry of lung slices and immunofluorescence of HLF culture. A: Immunohistochemistry of lung slicedemonstrating vimentin positive fibroblastsand vimentin negative airway smooth muscle cells. B: Immunohistochemistry of lung slicedemonstrating S100A4 positive fibroblastsand S100A4 negative airway smooth muscle cells. C: Immunofluorescence of HLF culture. D: Immunofluorescence of HLF culture. Supplemental figure 2: Demographic donor data of HLF cell lines. Supplemental figure 3: Dose response of treprostinil on TGF-β induced phosphorylation of SMAD3. Measurement of TGF-β-inducedphosphorylation of SMAD3 in HLF preincubated with treprostinil. Treprostinil has little effect on inhibiting TGF-β-induced phosphorylation of SMAD3. Supplemental figure 4: Dose response of IPR blocker on treprostinil phosphorylation of HSP20. HLF were pretreated with an IPR blocker, treated with treprostinil, then stimulated with TGF-βand phosphorylation of HSP20 was assessed.
Copyright
The authors
Creative Commons License
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Comments
This article was originally published in Respiratory Research, volume 26, in 2025. https://doi.org/10.1186/s12931-025-03387-3