Macropinocytosis inhibition attenuates profibrotic responses in lung fibroblasts and pulmonary fibrosis models
Ivan O. Rosas, Aaron K. McDowell-Sanchez, Santiago Sanchez, Juan D. Cala-Garcia, Alan R. Waich Cohen, Elisa Ruiz-Echartea, Scott A. Ochsner, Daniel C. Kraushaar, Lindsay J. Celada, Dandan Sun, Francesca Polverino, Cristian Coarfa, Neil J. McKenna, Konstantin Tsoyi
Ivan O. Rosas, Aaron K. McDowell-Sanchez, Santiago Sanchez, Juan D. Cala-Garcia, Alan R. Waich Cohen, Elisa Ruiz-Echartea, Scott A. Ochsner, Daniel C. Kraushaar, Lindsay J. Celada, Dandan Sun, Francesca Polverino, Cristian Coarfa, Neil J. McKenna, Konstantin Tsoyi
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Research Article Cell biology Pulmonology

Macropinocytosis inhibition attenuates profibrotic responses in lung fibroblasts and pulmonary fibrosis models

Abstract

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disorder with limited treatment options. Macropinocytosis is one of the key cellular processes involved in nutrient consumption from the extracellular environment under stress conditions. Here, we studied the role of macropinocytosis in experimental pulmonary fibrosis models. We found that macropinocytosis is increased in human lung fibroblasts (HLFs) derived from patients with IPF. The inhibition of macropinocytosis with 5-(n-ethyl-n-isopropyl)-amiloride (EIPA) inhibited profibrotic responses in IPF-derived and TGF-β1–stimulated HLFs and reduced pulmonary fibrosis in bleomycin-injured (Bleo-injured) mice. EIPA exerted its antifibrotic effects by regulating amino acid uptake, mammalian target of rapamycin complex 1 (mTORC1) activation and mesenchyme homeobox1 (MEOX1) expression in activated HLFs. Fittingly, genetic inhibition of macropinocytosis also ameliorated lung fibroblast activation and pulmonary fibrosis in mice. Using IPF-derived precision cut lung slices (PCLSs), we observed robust repression of profibrotic gene expression programs in EIPA-treated PCLSs across different fibroblast subpopulations. Finally, we found that imipramine (Imi), a tricyclic antidepressant approved by the FDA, effectively inhibited macropinocytosis and ameliorated profibrotic responses in lung fibroblasts, Bleo-injured mice, and IPF-derived PCLSs. Taken together, our results suggest that macropinocytosis inhibition can be considered as a potential therapeutic strategy to treat pulmonary fibrosis.

Authors

Ivan O. Rosas, Aaron K. McDowell-Sanchez, Santiago Sanchez, Juan D. Cala-Garcia, Alan R. Waich Cohen, Elisa Ruiz-Echartea, Scott A. Ochsner, Daniel C. Kraushaar, Lindsay J. Celada, Dandan Sun, Francesca Polverino, Cristian Coarfa, Neil J. McKenna, Konstantin Tsoyi

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Figure 1

Macropinocytosis is elevated in IPF-derived lung fibroblasts and its inhibition regulates profibrotic responses.

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Macropinocytosis is elevated in IPF-derived lung fibroblasts and its inh...
(A and B) Control and IPF-derived lung fibroblasts were conditioned in low-serum media for 24 hours, as described in Methods. 24 hours later, culture media was changed, and cells were incubated with FITC-dextran (70 kDa, 0.5 mg/mL) for 1 hour. Then, cells were washed 3 times with PBS. Macropinocytosis was measured by fluorescent microscopy (A) (scale bar: 25 μm) and flow cytometry (B) to measure fluorescence intensity of FITC-dextran in cells (n = 5 for A and n = 8 for B in each condition). (C and D) Cells were treated with vehicle (Veh) or EIPA (12.5 μM) for 24 hours. Then, cells were harvested and subjected to Western blot (C) and qRT-PCR (D) to measure collagen 1 and α-SMA expression, as described in Supplemental Methods and Supplemental Table 1 (n = 5 each condition). (E and F) Control HLFs were treated with EIPA (12.5 μM) with or without TGF-β1 (10 ng/mL) for 24 hours. Then, cells were harvested and subjected to Western blot (E) and qRT-PCR (F), as described in Methods (n = 4 each condition). (G) Control HLFs were mixed with collagen 1 solution, as described in Methods, and treated with EIPA (12.5 μM) with or without TGF-β1 (10 ng/mL). Gel size was measured at 0 and 24 hours after collagen gelation (n = 4 for each condition). Data are shown as the mean ± SEM. *P < 0.05, vs. unstimulated or Control + Veh; †P < 0.05, vs. IPF + Veh or TGF-β1 alone; significant comparisons by Student’s t test (A and B) or 1-way ANOVA (C–G).