Induced pluripotent stem cell therapy ameliorates hyperoxia-augmented ventilator-induced lung injury through suppressing the Src pathway

Abstract

Background: High tidal volume (VT) mechanical ventilation (MV) can induce the recruitment of neutrophils, release of inflammatory cytokines and free radicals, and disruption of alveolar epithelial and endothelial barriers. It is proposed to be the triggering factor that initiates ventilator-induced lung injury (VILI) and concomitant hyperoxia further aggravates the progression of VILI. The Src protein tyrosine kinase (PTK) family is one of the most critical families to intracellular signal transduction related to acute inflammatory responses. The anti-inflammatory abilities of induced pluripotent stem cells (iPSCs) have been shown to improve acute lung injuries (ALIs); however, the mechanisms regulating the interactions between MV, hyperoxia, and iPSCs have not been fully elucidated. In this study, we hypothesize that Src PTK plays a critical role in the regulation of oxidants and inflammation-induced VILI during hyperoxia. iPSC therapy can ameliorate acute hyperoxic VILI by suppressing the Src pathway.

Methods: Male C57BL/6 mice, either wild-type or Src-deficient, aged between 2 and 3 months were exposed to high VT (30 mL/kg) ventilation with or without hyperoxia for 1 to 4 h after the administration of Oct4/Sox2/Parp1 iPSCs at a dose of 5×10(7) cells/kg of mouse. Nonventilated mice were used for the control groups.

Results: High VT ventilation during hyperoxia further aggravated VILI, as demonstrated by the increases in microvascular permeability, neutrophil infiltration, macrophage inflammatory protein-2 (MIP-2) and plasminogen activator inhibitor-1 (PAI-1) production, Src activation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and malaldehyde (MDA) level. Administering iPSCs attenuated ALI induced by MV during hyperoxia, which benefited from the suppression of Src activation, oxidative stress, acute inflammation, and apoptosis, as indicated by the Src-deficient mice.

Conclusion: The data suggest that iPSC-based therapy is capable of partially suppressing acute inflammatory and oxidant responses that occur during hyperoxia-augmented VILI through the inhibition of Src-dependent signaling pathway.

Figure 1. Characterization of Oct4/Sox2/Parp1(OSP)-reprogrammed iPSCs.

(A) Parp1 is able to replace Klf-4 or c-Myc to generate mouse OSP-iPSCs cotransfected with Oct-4 and Sox-2. (B) Morphology of OSP-iPS cell colonies. (C) OSP-iPSC colonies were strongly positive for alkaline phosphatase stain. (D) The high passages of OSP-iPSCs were positive for SSEA-1 by immunofluorescent staining. Scale bars represent 200 µm (B & C) and 100 µm (D). DAPI = 4′, 6-diamidino-2-phenylindole; iPSC = induced pluripotent stem cell; OSP-iPSC = Oct4/Soc2/Parp1-reprogrammed induced pluripotent stem cell; SSEA-1 = stage-specific embryonic antigen 1.

Figure 2. iPSCs and Src-deficient mice suppressed hyperoxia-augmented lung stretch-induced Src phosphorylation.

(A, B) Western blot was performed using an antibody that recognizes the phosphorylated Src expression and an antibody that recognizes total Src expression from the lungs of nonventilated control mice and those subjected to V_T 30 ml/kg (V_T 30) with room air or hyperoxia at indicated time periods. Arbitrary units were expressed as the ratio of phospho-Src to Src (n = 5 per group). (C) Representative micrographs (x400) with phosphorylated Src staining of paraffin lung sections and quantification were from the lungs of nonventilated control mice and those subjected to V_T at 30 ml/kg for 4 h with room air or hyperoxia (n = 5 per group). iPSCs (5×10⁷ cells/kg, suspended in PBS) were injected via tail vein 1 h before mechanical ventilation. A dark-brown diaminobenzidine signal identified by arrows indicates positive staining for phospho-Src in the lung epithelium or interstitial, whereas shades of bluish tan signify nonreactive cells. *P<0.05 versus the nonventilated control mice with room air; †P<0.05 versus all other groups. Scale bars represent 20 µm. iPSC = induced pluripotent stem cell; O₂ = mice with hyperoxia; PBS = phosphate-buffered saline; RA = mice with room air; Src^+/− = Src deficient mice.

Figure 3. iPSCs and Src-deficient mice attenuated hyperoxia-augmented lung stretch-induced neutrophil sequestration, MIP-2 and PAI-1 production.

The effects of administering iPSCs or Src heterozygous knockout on (A) neutrophil infiltration, (B) MPO activity, (C) MIP-2, and (D) PAI-1 secretion in BAL fluid were from the lungs of nonventilated control mice and those subjected to V_T at 30 ml/kg for 4 h with room air or hyperoxia (n = 5 per group). iPSCs (5×10⁷ cells/kg, suspended in PBS) were injected via tail vein 1 h before mechanical ventilation. *P<0.05 versus the nonventilated control mice with room air; †P<0.05 versus all other groups. BAL = bronchoalveolar lavage fluid; MIP-2 = macrophage inflammatory protein-2; MPO = myeloperoxidase; PAI-1 = plasminogen activator inhibitor-1.

Figure 4. iPSCs and Src-deficient mice abrogated hyperoxia-augmented lung stretch-induced oxidative stress.

(A, B) Western blot was performed using antibodies that recognize NOX1 or NOX2 expression and an antibody that recognizes GAPDH expression from the lungs of nonventilated control mice and those subjected to V_T 30 ml/kg for 1 h with room air or hyperoxia. Arbitrary units were expressed as the ratio of NOX1 to GAPDH or NOX2 to GAPDH (n = 5 per group). (C) MDA level and (D) NADP⁺-to-NADPH ratio were from the lungs of nonventilated control mice and those subjected to V_T at 30 ml/kg for 4 h with room air or hyperoxia (n = 5 per group). iPSCs (5×10⁷ cells/kg, suspended in PBS) were injected via tail vein 1 h before mechanical ventilation. *P<0.05 versus the nonventilated control mice with room air; †P<0.05 versus all other groups. GAPDH = glyceraldehydes-phosphate dehydrogenase; MDA = malondialdehyde; NADP⁺ = nicotinamide adenine dinucleotide phosphate; NADPH = reduced NADP⁺; NOX1 = NADPH oxidase 1; NOX2 = NADPH oxidase 2.

Figure 5. iPSCs and Src-deficient mice reduced hyperoxia-augmented lung stretch-induced lung damage, microvascular leak, and lung edema.

(A) Histological examination (x200), (B) gross pathologic findings, (C) lung injury scores, (D) lung EBD, and (E) the wet-to-dry ratio were from the lungs of nonventilated control mice and those subjected to V_T at 30 ml/kg for 4 h with room air or hyperoxia (n = 5 per group). iPSCs (5×10⁷ cells/kg, suspended in PBS) were injected via tail vein 1 h before mechanical ventilation. *P<0.05 versus the nonventilated control mice with room air; †P<0.05 versus all other groups. EBD = Evans blue dye.

Figure 6. iPSCs and Src-deficient mice abrogated hyperoxia-augmented lung stretch-induced epithelial apoptosis and gas exchange.

Representative micrographs with (A) a transmission electron microscopic image (x6000), (B) TUNEL staining of paraffin section (x400), (C) quantitation, and (D) gas exchange (A, n = 3 per group; B, C, and D, n = 5 per group) were from the lungs of the control mice and those subjected to V_T at 30 ml/kg for 4 h with room air or hyperoxia. iPSCs (5×10⁷ cells/kg, suspended in PBS) were injected via tail vein 1 h before mechanical ventilation. Highly condensed and fragmented heterochromatin of bronchial epithelial cells indicates apoptosis. A dark-brown diaminobenzidine signal indicated positive staining of apoptotic cells, whereas shades of blue–green to greenish tan signified nonreactive cells. Apoptotic cells are identified by arrows. *P<0.05 versus the nonventilated control mice with room air; †P<0.05 versus all other groups. Scale bars represent 2 or 20 µm. A-aDO₂ = alveolar-arterial oxygen difference; TUNEL = terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling.