Mitochondrial Transplantation Ameliorates Pulmonary Fibrosis by Suppressing Myofibroblast Activation

Abstract

Idiopathic pulmonary fibrosis (IPF) is a pulmonary disease characterized by excessive extracellular matrix protein deposition in the lung interstitium, subsequently causing respiratory failure. IPF still has a high medical unmet requirement due to the lack of effective treatments to inhibit disease progression. The etiology of IPF remains unclear, but mitochondrial dysfunction is considered to be associated with IPF development. Therefore, targeting mitochondrial abnormalities would be a promising strategy for treating IPF. Recently, exogenous mitochondrial transplantation has been beneficial for treating mitochondrial dysfunction. The current study aimed to examine the therapeutic effect of mitochondrial transplantation on IPF in vitro and in vivo. Mitochondria were isolated from human umbilical cord mesenchymal stem cells, referred to as PN-101. Human lung fibroblasts and human bronchial epithelial cells were exposed to transforming growth factor-β, followed by PN-101 treatment to determine the in vitro efficacy of mitochondrial transplantation. An IPF mouse model established by a single intratracheal instillation of bleomycin was utilized to determine the in vivo efficacy of the intravenously treated mitochondria. PN-101 attenuated mitochondrial damage, inhibited EMC production, and suppressed epithelial-to-mesenchymal transition in vitro. Additionally, intravenous PN-101 administration alleviated bleomycin-induced fibrotic processes in the IPF mouse model with a therapeutic context. Our data indicate that PN-101 is a novel and potential therapeutic agent for IPF.

Keywords: anti-inflammation; antiapoptosis; idiopathic pulmonary fibrosis (IPF); mitochondria; stem cell; transplantation.

Figure 1

Exogenous mitochondrial transfer to human lung cells and reversal of TGF-β-induced mitochondrial dysfunction. (A) Schematic diagram of PN-101^dsRED treatment into human lung fibroblasts (CCD8-Lu) and human bronchial epithelial cells (HBECs). PN-101^dsRED was obtained from UC-MSCs, which were infected with a lentivirus expressing MTS-dsRED. (B) Representative immunofluorescence images. The red colored dots indicate the exogenously transferred PN-101^dsRED. The cell nuclei were stained with DAPI. PN-101^dsRED was successfully transferred into the CCD8-Lu cells and HBECs (white arrows). The scale bar indicates 10 μm. (C) Schematic representation of PN-101 treatment in the TGF-β-induced fibrosis model. CCD8-Lu cells and HBECs were seeded, starved in a culture medium without FBS for 24 h, and then treated with TGF-β at 2.5 ng/mL. After 6 h, various PN-101 concentrations were administered for 24 h. Intracellular ATP and mitochondrial membrane potential (MtMP) were measured in CCD8-Lu cells (D,E) and HBECs (F,G), respectively. PN-101 treatment significantly attenuated the TGF-β-induced decrease in ATP and MtMP levels. All data are presented as mean ± SEM. * p < 0.05 and *** p < 0.001 versus control and ^# p < 0.05 and ^### p < 0.001 vs. PN-101 non-treatment.

Figure 2

Anti-fibrotic effects of mitochondrial transplantation in the TGF-β-induced CCD8-Lu cells. Human lung fibroblasts, CCD8-Lu cells, were treated with TGF-β for 6 h after PN-101 administration for 24 h. (A–D) The mRNA expression of fibrotic genes was measured with quantitative RT-PCR. PN-101 treatment attenuated the mRNA expression of fibronectin, α-SMA, NOX4, and CTGF in TGF-β-stimulated CCD8-Lu cells dose-dependently. (E–H) Collagen type 1 A1 (Col1A1), fibronectin, and α-SMA protein levels were measured with a Western blot assay. PN-101 attenuated Col1A1, fibronectin, and α-SMA protein expression in TGF-β-stimulated CCD8-Lu cells in a concentration-dependent manner. (I) The Col1A1 and α-SMA proteins were highly expressed in TGF-β-stimulated CCD8-Lu cells as measured using an immunocytochemistry assay, whereas PN-101 abolished the TGF-β-stimulated expression of the Col1A1 (J) and α -SMA proteins (K). The scale bar indicates 10 μm. Data are presented as the mean ± SEM (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. PN-101 non-treatment.

Figure 3

Anti-fibrotic effects of stem cell-derived mitochondria. Anti-fibrotic effects of UC-MSC-derived mitochondria (PN-101) were compared with those of HEK293-derived mitochondria (hkMT). (A–D) The mRNA expression of fibrotic genes was assessed by qRT-PCR. PN-101 mitigated the mRNA expression of fibronectin, α -SMA, NOX4, and CTGF in TGF-β-stimulated CCD8-Lu cells, whereas hkMT did not. (E–H) Col1A1, fibronectin, α-SMA, and CTGF protein levels were assessed with a Western blot assay. PN-101, but not hkMT, attenuated fibrotic protein expression in TGF-β-stimulated CCD8-Lu cells. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. control. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 vs. mitochondria non-treatment.

Figure 4

Epithelial-to-mesenchymal transition suppression by PN-101 in human bronchial epithelial cells. Human bronchial epithelial cells (HBECs) were treated with TGF-β of 2.5 ng/mL for 6 h after PN-101 administration for 24 h. (A–D) Fibronectin, vimentin, and α-SMA protein levels were evaluated by a Western blot assay. PN-101 attenuated fibronectin, vimentin, and α-SMA protein expression in TGF-β-stimulated HBECs. (E) An ICC assay demonstrated that PN-101 suppressed TGF-β-stimulated fibronectin, vimentin, and α -SMA protein expression in HBECs. The scale bar indicates 10 μm. Data are presented as the mean ± SEM (n = 3). ** p < 0.01 and *** p < 0.001 vs. control. ^# p < 0.05, and ^## p < 0.01 vs. PN-101 non-treatment.

Figure 5

Effect of mitochondrial transplantation on body weight and lung weight. (A) Schematic representation of PN-101 administration to BLM-induced mice. The mice were intratracheally instilled with BLM of 0.33 mpk and intravenously injected three times with 2.5-μg PN-101/head (0.125 mpk) or 5-μg PN-101/head (0.25 mpk) 7 days after the BLM treatment. (B,C) BLM instillation decreased body weight and was restored to normal with PN-101 treatment. (D,E) BLM treatment increased lung wet weight even when expressed relative to body weight. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus sham. ^# p < 0.05 and ^## p < 0.01 vs. PN-101 non-treatment.

Figure 6

Attenuation of fibrotic processes by mitochondrial transplantation in BLM-induced PF mice. (A) Representative lung tissue sections stained with H&E. The sections in the sham group exhibited normal morphology, whereas those of the BLM group demonstrated structural disorder and alveolar septum widening. PN-101 treatment significantly improved these histopathological lesions. Scale bars indicate 100 μm. (B) The Ashcroft fibrosis score demonstrated that PN-101 significantly alleviated PF in BLM-treated mice. (C) The effect of PN-101 on total cell counts in the BALF of the BLM-induced PF mice. (D,E) The effect of PN-101 on the number of macrophages and lymphocytes in the BALF. Data are presented as the mean ± SD (n = 5). *** p < 0.001 vs. sham group. ^# p < 0.05 and ^## p < 0.01 vs. PN-101 non-treatment.