The main e-cigarette component vegetable glycerin enhances neutrophil migration and fibrosis in endotoxin-induced lung injury via p38 MAPK activation.

We investigated the effects of vegetable glycerin (VG), a main e-cigarette constituent, on endotoxin-induced acute lung injury (ALI). Mice received intratracheal administration of 30% VG in phosphate buffered saline (PBS) vehicle or only PBS (control) for 4 days. On Day 5, mice received an intratracheal instillation of lipopolysaccharide (LPS) (LPS group and VG + LPS group) or PBS (VG group and control group). Lung histopathology, expression of chemokine receptors, and regulatory signaling were analyzed 24 h after the Day 5 treatment. VG significantly increased ALI-associated histopathological and fibrotic changes in both the VG group and LPS-induced ALI mice (VG + LPS group). Immunohistochemistry (IHC) and western blot analyses revealed that VG administration resulted in upregulation of neutrophil markers [lymphocyte antigen 6 complex locus G6D (Ly6G) and myeloperoxidase (MPO)] as well as upregulation of the expression of transforming growth factor-ß (TGF-ß), a central mediator of fibrogenesis, in the lungs of both VG and VG + LPS groups. VG enhanced the expression of adhesion molecules [very late antigen 4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1)] and increased activation of p38 mitogen-activated protein kinase (p38 MAPK) to prompt neutrophil recruitment in the lungs of mice with ALI. Intraperitoneal administration of a p38 inhibitor attenuated these histopathological changes significantly as well as VG-induced upregulation in expression of Ly6G, MPO, VLA-4, VCAM-1, TGF-ß, and collagen-1 in mice with ALI. In conclusion, VG enhances neutrophil chemotaxis and fibrosis and it amplifies the inflammatory response associated with LPS-induced ALI in the lungs via enhancement of p38 MAPK activity.

Nintedanib Inhibits Endothelial Mesenchymal Transition in Bleomycin-Induced Pulmonary Fibrosis via Focal Adhesion Kinase Activity Reduction.

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease (ILD). Pulmonary fibroblasts play an important role in the development of IPF. Emerging evidence indicates that pulmonary endothelial cells could be the source of pulmonary fibroblasts through endothelial mesenchymal transition (EndoMT), which contributes to pulmonary fibrosis. EndoMT is a complex process in which endothelial cells lose their expression of endothelial markers and give rise to the characteristics of mesenchymal cells, including morphological fibroblast-like change and the expression of mesenchymal markers, which result in cardiac, renal, and dermal fibroses. Furthermore, EndoMT inhibition attenuates pulmonary fibrosis. Herein, we demonstrate that nintedanib, a tyrosine kinase receptor inhibitor, ameliorated murine bleomycin (BLM)-induced pulmonary fibrosis and suppressed the in vivo and in vitro models of EndoMT. We demonstrated that the activity of focal adhesion kinase (FAK), a key EndoMT regulator, increased in murine lung tissues and human pulmonary microvascular endothelial cells after BLM stimulation. Nintedanib treatment inhibited BLM-induced FAK activation and thus suppressed both in vivo and in vitro BLM-induced EndoMT. Importantly, we found that the VEGF/FAK signaling pathway was involved in nintedanib regulating EndoMT. These novel findings help us understand the mechanism and signaling pathway of EndoMT to further develop more efficacious drugs for IPF treatment.

Nintedanib Regulates GRK2 and CXCR2 to Reduce Neutrophil Recruitment in Endotoxin-Induced Lung Injury.

The role of nintedanib, a multiple tyrosine kinase inhibitor, in the treatment of sepsis-induced acute lung injury (ALI) remains unclear. Lipopolysaccharide (LPS), also known as endotoxin, has been used to induce ALI. The goal of this study was to assess the effect of nintedanib in attenuating the histopathological changes of LPS-induced ALI. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h and 10 min before intratracheal endotoxin instillation. Lung histopathological characteristics, adhesion molecule expression, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed after 24 h. We found that nintedanib significantly reduced histopathological changes and neutrophil recruitment in LPS-induced ALI. The number of neutrophils in bronchoalveolar lavage fluid (BALF) was reduced in nintedanib-treated relative to untreated mice with ALI. Nintedanib mediated the downregulation of the chemotactic response to LPS by reducing the expression of adhesion molecules and the phosphorylated p38:total p38 mitogen-activated protein kinase (MAPK) ratio in the lungs of mice with ALI. Nintedanib also reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly6G) and very late antigen 4 (VLA-4) in BALF neutrophils and mediated the downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in mice with LPS-induced ALI. Nintedanib improved the histopathological changes of LPS-induced ALI by reducing neutrophil chemotaxis. These effects were mediated by the inhibition of adhesion molecules via the activation of GRK2 and the inhibition of p38 MAPK and CXCR2.

Induced Pluripotent Stem Cell-Derived Conditioned Medium Promotes Endogenous Leukemia Inhibitory Factor to Attenuate Endotoxin-Induced Acute Lung Injury.

The conditioned medium of induced pluripotent stem cells (iPSC-CM) can attenuate neutrophil recruitment and endothelial leakage of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Therefore, we investigated the mechanisms by which iPSC-CM regulate the interaction between neutrophils and the endothelium in ALI. Murine iPSCs (miPSCs) were delivered intravenously to male C57BL/6 mice (8-12 weeks old) 4 h after intratracheal LPS injection. A miPSC-derived conditioned medium (miPSC-CM) was delivered intravenously to mice after intratracheal LPS injection. DMSO-induced HL-60 cells (D-HL-60, neutrophil-like cells) and human umbilical vein endothelial cells (HUVECs) were used as in vitro models to assess the interaction of neutrophils and endothelial cells. miPSC-CM diminished the histopathological changes in the lungs and the neutrophil count in bronchoalveolar lavage fluids of ALI mice. miPSC-CM attenuated the expression of adhesion molecules in the lungs of ALI mice. Human iPSC conditioned medium (hiPSC-CM) reduced the expression of adhesion molecules in a HUVEC and D-HL-60 co-culture after LPS stimulation, which decreased the transendothelial migration (TEM) of D-HL-60. A human angiogenesis factors protein array revealed that leukemia inhibitory factor (LIF) was not detected in the absence of D-HL-60 and hiPSC-CM groups. hiPSC-CM significantly promoted the production of endogenous LIF in in vitro models. Administration of an anti-LIF antibody not only reversed the effect of iPSC-CM in ALI mice, but also blocked the effect of iPSC-CM on neutrophils TEM in in vitro models. However, a controlled IgG had no such effect. Our study demonstrated that iPSC-CM promoted endogenous LIF to inhibit neutrophils TEM and attenuate the severity of sepsis-induced ALI.

Nintedanib Reduces Neutrophil Chemotaxis via Activating GRK2 in Bleomycin-Induced Pulmonary Fibrosis.

Neutrophils are involved in the alveolitis of idiopathic pulmonary fibrosis (IPF). However, their pathogenic mechanisms are still poorly understood. Nintedanib has antifibrotic and anti-inflammatory activity in IPF. This study aimed to investigate the regulatory mechanism of nintedanib on neutrophil chemotaxis in bleomycin (BLM)-induced pulmonary fibrosis. Nintedanib was administered via oral gavage to male C57BL/6 mice 24 h after a bleomycin intratracheal injection (1.5 U/kg). Lung histopathological findings, the expression of cytokines, and the regulatory signaling pathways of neutrophil chemotaxis were analyzed. The effect of nintedanib was also investigated in a mouse model with adoptive neutrophil transfer in vivo. Nintedanib significantly decreased the histopathological changes and neutrophil recruitment in BLM-induced pulmonary fibrosis. Nintedanib mediated a downregulation of chemokine (C-X-C motif) receptor 2 (CXCR2) and very late antigen 4 (VLA-4) expression, as well as an upregulation of G protein-coupled receptor kinase 2 (GRK2) activity in peripheral blood neutrophils in BLM-induced pulmonary fibrosis. Nintedanib also decreased the activation of endothelial cells by the decreased expression of vascular cell adhesion molecule 1 (VCAM-1). The effect of nintedanib on regulating neutrophil chemotaxis was also confirmed by a mouse model with adoptive neutrophil transfer in vivo. In conclusion, nintedanib reduces neutrophil chemotaxis and endothelial cell activation to regulate the severity of BLM-induced pulmonary fibrosis. These effects are associated with an enhancement of GRK2 activity and a reduction in CXCR2 and VLA-4 expression on neutrophils and a decrease in VCAM-1 expression on endothelial cells.