Exosomes from adipose tissue-derived mesenchymal stem cells ameliorate histone-induced acute lung injury by activating the PI3K/Akt pathway in endothelial cells

Abstract

Background: Mesenchymal stem cells (MSCs), including adipose-derived mesenchymal stem cells (ADSCs), have been shown to attenuate organ damage in acute respiratory distress syndrome (ARDS) and sepsis; however, the underlying mechanisms are not fully understood. In this study, we aimed to explore the potential roles and molecular mechanisms of action of ADSCs in histone-induced endothelial damage.

Methods: Male C57BL/6 N mice were intravenously injected with ADSCs, followed by histones or a vehicle. The mice in each group were assessed for survival, pulmonary vascular permeability, and histological changes. A co-culture model with primary human umbilical vein endothelial cells (HUVECs) exposed to histones was used to clarify the paracrine effect of ADSCs. Overexpression and inhibition of miR-126 ADSCs were also examined as causative factors for endothelial protection.

Results: The administration of ADSCs markedly improved survival, inhibited histone-mediated lung hemorrhage and edema, and attenuated vascular hyper-permeability in mice. ADSCs were engrafted in the injured lung and attenuated histone-induced endothelial cell apoptosis. ADSCs showed endothelial protection (via a paracrine effect) and Akt phosphorylation in the histone-exposed HUVECs. Notably, increased Akt phosphorylation by ADSCs was mostly mediated by exosomes in histone-induced cytotoxicity and lung damage. Moreover, the expression of miR-126 was increased in exosomes from histone-exposed ADSCs. Remarkably, the inhibition of miR-126 in ADSCs failed to increase Akt phosphorylation in histone-exposed HUVECs.

Conclusion: ADSC-derived exosomes may exert protective effects on endothelial cells via activation of the PI3K/Akt pathway.

Keywords: Acute lung injury; Acute respiratory distress syndrome; Adipose-derived mesenchymal stem cells; Endothelial damage; Exosomes; Histones; PI3K/Akt signaling pathway; Sepsis; miR-126.

Fig. 1

Effects of histones with or without ADSC administration in mice. a Representative pictures of the lung CT scan 1 h after injection of 75 mg/kg histones. b Kaplan-Meier survival curves showing survival in the saline+histones group and the ADSCs+histones group. N = 8 in each group. ^*p = 0.0256 vs. saline+histones group. c In vivo pulmonary vascular permeability assay measured by Evans Blue dye. N = 5 in each group. ^**p < 0.01 vs. control group, ^†p < 0.05 vs. saline+histones group. d Representative photomicrographs of lung sections with H&E staining 6 h after injections of ADSCs and histones. Scale Bar, 50 μm

Fig. 2

Immunofluorescent images in lung tissues. Cleaved caspase-3 (green, arrows) and endothelial cells are stained with anti-CD31 antibody (red). Overlap images indicate that the cleaved form of caspase-3 is present in endothelial cells. Nuclei were counterstained with Hoechst 33258 dye (blue)

Fig. 3

Fluorescent tracing of infused ADSCs. a Detection of red-fluorescent-labeled ADSCs using CellVue Claret Far Red Fluorescent Cell Linker Kits by fluorescence microscopy. Nuclei were counterstained with Hoechst 33258 dye (blue). b Cell Linker-labeled ADSCs were detected in the lung with or without histones. c Overlap images indicate that the cleaved form of caspase-3 (green) is present with Cell Linker-labeled ADSCs

Fig. 4

Paracrine effect of ADSCs against histone-mediated cytotoxicity with HUVECs. a The relative cell viability of HUVECs after co-culture with or without ADSCs-DMSO or ADSCs-GW4869 exposed to histones (100 μg/mL) for 4 h. N = 6 in each group. ^**p < 0.01 vs. control, ^†p < 0.05 vs. histones. b–d Representative immunoblots and densitometric analysis of phosphorylated Akt and cleaved caspase-3 in HUVECs 4 h after histone challenge with or without ADSCs-DMSO and ADSCs-GW4869. N = 4 in each group. ^*p < 0.05 vs. control, ^†p < 0.05 vs. histones, ^‡p < 0.05 vs. histones+ADSCs-DMSO

Fig. 5

Inhibition of LY294002 and GW4869 on the protective effect of ADSCs in mice. a, b Representative immunoblots and densitometric analysis of phosphorylated Akt in lung tissue 6 h after histone challenge with or without ADSCs, ADSCs-GW4869, and LY294002. N = 5 in each group. ^**p < 0.01 vs. control group, ^†p < 0.05 vs. ADSCs+histones group. c Kaplan-Meier survival curves showing survival in the saline+histones group, the ADSCs+histones group, the GW4869 ADSCs+histones group, and the LY294002 + ADSCs+histones group. N = 8 in each group. d In vivo pulmonary vascular permeability assay measured by Evans Blue dye. N = 5 in each group. ^**p < 0.01 vs. control group, ^†p < 0.05 vs. saline+histones group, ^††p < 0.01 vs. saline+histones group, ^‡‡p < 0.01 vs. ADSCs+histones group. e Representative photomicrographs of lung sections with H&E staining 6 h after injections of ADSCs or ADSCs-GW4869 and histones with or without LY294002. Scale Bar, 50 μm

Fig. 6

Inhibition of miR-126 on the protective effect of ADSCs against histone-mediated cytotoxicity with HUVECs. a, b The expression of miR-126 in ADSCs and exosomes before or after 2 h of exposure to histones was evaluated using RT-PCR. N = 3 in each group. ^*p < 0.05, ^**p < 0.01 vs. control group. c The relative cell viability of histone-exposed HUVECs (100 μg/mL, 4 h) after co-culture with ADSCs, miR-126 mimics ADSCs or miR-126 inhibitor ADSCs. N = 3 in each group. ^**p < 0.01 vs. control, ^††p < 0.01 vs. miR-126 mimic, ^‡p < 0.05 vs. control. d–f Immunoblot results of phosphorylated Akt and cleaved capsase-3 in histone-exposed HUVECs (100 μg/mL, 4 h) co-cultured with ADSCs, miR-126 mimic ADSCs or miR-126 inhibitor ADSCs. N = 4~6 in each group. ^*p < 0.05, ^**p < 0.01 vs. control, ^††p < 0.01 vs. miR-126 mimic