Cyclic Phytosphingosine-1-Phosphate Primed Mesenchymal Stem Cells Ameliorate LPS-Induced Acute Lung Injury in Mice.

Background and Objectives: O-cyclic phytosphingosine-1-phosphate (cP1P) is a synthetic chemical and has a structure like sphingosine-1-phosphate (S1P). S1P is known to promote cell migration, invasion, proliferation, and anti-apoptosis through hippocampal signals. However, S1P mediated cellular-, molecular mechanism is still remained in the lung. Acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) are characterized by excessive immune response, increased vascular permeability, alveolar-peritoneal barrier collapse, and edema. In this study, we determined whether cP1P primed human dermal derived mesenchymal stem cells (hdMSCs) ameliorate lung injury and its therapeutic pathway in ALI mice. Methods and Results: cP1P treatment significantly stimulated MSC migration and invasion ability. In cytokine array, secretion of vascular-related factors was increased in cP1P primed hdMSCs (hdMSCcP1P), and cP1P treatment induced inhibition of Lats while increased phosphorylation of Yap. We next determined whether hdMSCcP1P reduce inflammatory response in LPS exposed mice. hdMSCcP1P further decreased infiltration of macrophage and neutrophil, and release of TNF-α, IL-1ß, and IL-6 were reduced rather than naïve hdMSC treatment. In addition, phosphorylation of STAT1 and expression of iNOS were significantly decreased in the lungs of MSCcP1P treated mice. Conclusions: Taken together, these data suggest that cP1P treatment enhances hdMSC migration in regulation of Hippo signaling and MSCcP1P provide a therapeutic potential for ALI/ARDS treatment.

Establishment of a human induced pluripotent stem cell derived alveolar organoid for toxicity assessment.

Alveolar epithelial cells (AECs) are vulnerable to injury, which can result in epithelial hyperplasia, apoptosis, and chronic inflammation. In this study, we developed human induced pluripotent stem cell (hiPS) cell-derived AECs (iAECs) and the iAECs based organoids (AOs) for testing AEC toxicity after chemical exposure. HiPS cells were cultured for 14 days with differentiation medium corresponding to each step, and the iAECs-based AOs were maintained for another 14 days. SFTPC and AQP5 were expressed in the AOs, and mRNA levels of SOX9, NKX2.1, GATA6, HOPX, and ID2 were increased. The AOs were exposed for 24 h to nine chemical substances, and IC50 values of the nine chemicals were determined using MTT assay. When the correlations between iAECs 2D culture and AOs 3D culture were calculated using Pearson's correlation coefficient r value, the nine chemicals that caused a significant decrease of cell viability in 3D culture were found to be highly correlated in 2D culture. The cytotoxicity and nitric oxide release in AO cultured with macrophages were then investigated. When AOs with macrophages were exposed to sodium chromate for 24 h, the IC50 value and nitric oxide production were higher than when the AOs were exposed alone. Taken together, the AO-based 3D culture system provides a useful platform for understanding biological characteristics of AECs and modeling chemical exposures.

L-carnosine Attenuates Bleomycin-Induced Oxidative Stress via NFκB Pathway in the Pathogenesis of Pulmonary Fibrosis.

Idiopathic Pulmonary fibrosis (IPF), a chronic interstitial lung disease, has pulmonary manifestations clinically characterized by collagen deposition, epithelial cell injury, and a decline in lung function. L-carnosine, a dipeptide consisting of ß-alanine and L-histidine, has demonstrated a therapeutic effect on various diseases because of its pivotal function. Despite the effect of L-carnosine in experimental IPF mice, its anti-oxidative effect and associated intercellular pathway, particularly alveolar epithelial cells, remain unknown. Therefore, we demonstrated the anti-fibrotic and anti-inflammatory effects of L-carnosine via Reactive oxygen species (ROS) regulation in bleomycin (BLM)-induced IPF mice. The mice were intratracheally injected with BLM (3 mg/kg) and L-carnosine (150 mg/kg) was orally administrated for 2 weeks. BLM exposure increased the protein level of Nox2, Nox4, p53, and Caspase-3, whereas L-carnosine treatment suppressed the protein level of Nox2, Nox4, p53, and Caspase-3 cleavage in mice. In addition, the total SOD activity and mRNA level of Sod2, catalase, and Nqo1 increased in mice treated with L-carnosine. At the cellular level, a human fibroblast (MRC-5) and mouse alveolar epithelial cell (MLE-12) were exposed to TGFß1 following L-carnosine treatment to induce fibrogenesis. Moreover, MLE-12 cells were exposed to cigarette smoke extract (CSE). Consequently, L-carnosine treatment ameliorated fibrogenesis in fibroblasts and alveolar epithelial cells, and inflammation induced by ROS and CSE exposure was ameliorated. These results were associated with the inhibition of the NFκB pathway. Collectively, our data indicate that L-carnosine induces anti-inflammatory and anti-fibrotic effects on alveolar epithelial cells against the pathogenesis of IPF.

Reduced receptor for advanced glycation end products is associated with α-SMA expression in patients with idiopathic pulmonary fibrosis and mice.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease. Despite alveolar epithelial cells is crucial role in lung, its contribution and the associated biomarker remain unknown in the pathogenesis of IPF. Recently, environmental factors including stone dust, silica and cigarette smoking were found as risk factors involved in IPF. Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super family of cell surface receptors. It has been shown that interaction between RAGE and its ligands on immune cells mediates cellular migration and regulation of pro-inflammation. RAGE is highly expressed in the lung, in particular, alveolar epithelial cells. Therefore, we determined whether RAGE expression is associated with fibrosis-associated genes in patients with IPF and mice. RESULTS: When bleomycin (BLM) was intratracheally administered to C57BL/6 mice for 1, 2 weeks, macrophage and neutrophils were significantly increased. The fibrotic nodule formed and accumulation of collagen was determined after BLM injection in H&E- and Masson's trichrome staining. Levels of elastin, Col1a1 and fibronectin were increased in quantitative real-time PCR and protein levels of α-SMA was increased in western blot analysis. In the lung tissues of 1 mg/kg BLM-induced mice, RAGE expression was gradually decreased in 1- and 2 weeks in immunohistochemistry and western blot analysis, and 3 mg/kg of BLM-induced mice exhibited decreased RAGE levels while α-SMA expression was increased. We next determined RAGE expression in the lungs of IPF patients using immunohistochemistry. As a result, RAGE expression was decreased, while α-SMA expression was increased compared with non-IPF subjects. CONCLUSIONS: Our findings suggest that reduced RAGE was associated with increased fibrotic genes in BLM-induced mice and patients with IPF. Therefore, RAGE could be applied with a biomarker for prognosis and diagnosis in the pathogenesis of IPF.