Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to mechanical ventilation-induced pulmonary fibrosis

Background: Mechanical ventilation (MV) can induce pulmonary fibrosis. This study aims to investigate whether MV-induced pulmonary fibrosis is associated with aerobic glycolysis and seeks to uncover the underlying mechanisms mediated by integrin β3-pyruvate kinase M2 (PKM2) pathway. Methods: PKM2 knockdown or inhibition, integrin β3 knockout or inhibition and wild-type mice were exposed to MV (20 mL/kg) for 2 h. Results: Mice exposed to MV exhibited increased expression of collagen deposition, and upregulation of α-smooth muscle actin and collagen I in lung tissues. Single cells analysis showed that MV-induced pulmonary fibrosis was associated with increased gene expression of integrin and glycolysis in pulmonary fibroblasts, as well as upregulation of glycolytic products tested by metabolomics. Meanwhile, increased protein level of integrin β3 and PKM2 was confirmed by western blot and immunohistochemistry. Double immunofluorescence staining and flow cytometric analysis showed increased number of fibronectin+/integrin β3+ and fibronectin+/PKM2+ fibroblasts in lung tissues. Furthermore, MV-induced aerobic glycolysis and pulmonary fibrosis were ameliorated after treatment with PKM2 knockdown-AAV and inhibition, or in integrin β3 knockout and inhibition mice. Conclusions: Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to MV-induced pulmonary fibrosis. The inhibition of aerobic glycolysis targeting integrin β3-PKM2 pathway may be a promising treatment for MV-induced pulmonary fibrosis.

. MV model and animal procedures. A, mice in the MV group were mechanically ventilated for 2 h using FiO2 0.21, VT: 20 ml/kg and respiratory rate 70 bpm, while mice in the sham group breathed spontaneously after intubation. B, intratracheal injection of PKM2 knockdown adeno-associated virus (AAV) and vector-AAV. C, gastric infusion of PKM2 inhibitor shikonin 10 mg/kg were administered to inhibit PKM2 daily for 3 days before MV. D, Integrin β3 knockout mice were randomly divided into sham and MV groups. E, Cilengitide, as integrin β3 inhibitor, was intraperitoneally administered daily for 3 days before MV, at a dose of 4 mg/kg.

Figure S5. PKM2 knockdown alleviates aerobic glycolysis and MV-induced pulmonary fibrosis.
Mice were treated intratracheally with vector-AAV or shPKM-AAV 4 weeks before mechanical ventilation (MV), for the duration of 2 h. Lung tissues were harvested 7 days after intubation. A, lung fibrosis was quantified by Masson's trichrome staining. Original magnification × 10, Scale bars correspond to 2.5 mm, n = 6, 3, 6, 5, respectively in each group. B, lung tissues were stained with fluorophorelabeled antibodies against fibroblast marker fibronectin and collagen I, 4',6-diamidino-2phenylindole stain was used to detect nuclei. Integrated optical density (IOD) of the specific fluorescent protein was analyzed by the Aipathwell to analyze the expression for the single or double stained fluorescent proteins.

Figure S6. PKM2 inhibitor alleviates aerobic glycolysis and MV-induced pulmonary fibrosis.
Mice were treated with PKM2 inhibitor shikonin 10 mg/kg in 100 ul corn oil daily for 3 consecutive days before being subjected to MV for 2 h. Lung tissues were harvested 7 days after intubation. A, lung fibrosis was accessed by Masson's trichrome staining, Original magnification × 10, Scale bars correspond to 2.5 mm, n = 6, 3, 6, 3, respectively in each group. B, lung tissues were stained with fluorophore-labeled antibodies against fibroblast marker fibronectin and collagen I, 4',6-diamidino-2phenylindole stain was used to detect nuclei. Integrated optical density (IOD) of the specific fluorescent protein was analyzed by the Aipathwell to analyze the expression for the single or double stained fluorescent proteins.

Figure S7. Integrin β3 inhibition ameliorates PKM2-mediated aerobic glycolysis and
MV-induced pulmonary fibrosis. A, Both wild-type and integrin β3-deficient (β3 +/-) mice were subjected to mechanical ventilation (MV) for 2 h. Lung tissues were harvested at day 7. Lung fibrosis was quantified by Masson's trichrome staining, original magnification × 10, scale bars correspond to 2.5 mm, n = 6, 5, 6, 6, respectively in each group. B, mice were treated intraperitoneally with integrin β3 inhibitor cilengitide 4 mg/kg once a day for 3 consecutive days before being subjected to MV for 2 h. Lung tissues were harvested 7 days after intubation. lung fibrosis was quantified by Masson's trichrome staining, original magnification × 10, scale bars correspond to 2.5 mm, n = 6 per group.

Figure S8. Integrin β3 inhibition ameliorates PKM2-mediated aerobic glycolysis and
MV-induced pulmonary fibrosis. A, B, both wild-type and integrin β3-deficient (β3 +/-) mice were subjected to mechanical ventilation (MV) for 2 h. Lung tissues were harvested at day 7. Lung tissues were stained with fluorophorelabeled antibodies against fibroblast marker fibronectin, pyruvate kinase M2(PKM2)and collagen I, 4',6-diamidino-2-phenylindole (DAPI) stain was used to detect nuclei. C,D, mice were treated intraperitoneally with integrin β3 inhibitor cilengitide 4mg/kg once a day for 3 consecutive days before being subjected to MV for 2 h. Lung tissues were harvested 7 days after intubation. Lung tissues were stained with fluorophore-labeled antibodies against fibroblast marker fibronectin, PKM2 and collagen I, DAPI stain was used to detect nuclei. Integrated optical density (IOD) of the specific fluorescent protein was analyzed by the Aipathwell to analyze the expression for the single or double stained fluorescent proteins.