Int J Stem Cells.  2023 May;16(2):191-201. 10.15283/ijsc23001.

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

• Park Y
• Jang J
• Lee J
• Baek H
• Park J
• Cha SR
• Lee SB
• Na S
• Kwon JW
• Hong SH
• Yang SR

• KMID: 2542832
• DOI: http://doi.org/10.15283/ijsc23001

Abstract

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 (hdMSC^cP1P ), and cP1P treatment induced inhibition of Lats while increased phosphorylation of Yap. We next determined whether hdMSC^cP1P reduce inflammatory response in LPS exposed mice. hdMSC^cP1P further decreased infiltration of macrophage and neutrophil, and release of TNF-α, IL-1β, 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 MSC^cP1P treated mice.
Conclusions
Taken together, these data suggest that cP1P treatment enhances hdMSC migration in regulation of Hippo signaling and MSC^cP1P provide a therapeutic potential for ALI/ARDS treatment.

Keyword

cP1P; MSC; ALI; HIPPO; STAT1

Figure

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  Fig. 1 An analogous compound of S1P, cP1P does not have cellular toxicity. (A) Structural formula of S1P and cP1P. (B) Cell viability was qua-ntified by MTT assay at various concentrations (Vehicle, 0.5, 1, 5 μM). All data are representative of multiple experiments. CTL: control group, ns: not significant.

• 

  Fig. 2 hdMSCcP1P was promoted homing by upregulated migration and invasion. (A) hdMSCs scratched by 1 ml tips and then treated to cP1P (0.5 μM, 1 μM, 5 μM) for 24 h. Scratch point captured by microscope (magnification ×40, Scale bars, 100 μm). (B) Wound healing degree of MSC was quantified by migration assay. After 24 h, various concentration was measured and presented as fold change. (C) Invasion assay was measured by trans well. Various concentrations. Original magnification, ×40. Scale bars, 100 μm. Cell numbers of various concentrations was counted and presented as numbers. (D) gene expression level of MMP2 and MMP9 was measured by real-time PCR. All data are representative of multiple experiments. CTL: control group, ns: not significant. *p<0.05, **p<0.01, and ***p<0.001 vs. control group.

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  Fig. 3 Changes in cytokines secreted by cP1P in hdMSC. (A) Heat map of hdMSCcP1P and non-primed hdMSC. Changes of cytokines were measured by cytokine assay kits. (B) Comparison of mean pixel density measurement between hdMSC and cP1P-primed hdMSC. (C) 1 μM cP1P-primed hdMSC and non-primed hdMSC measured mRNA expression level of hippo signaling pathway and migration associated factors by Real-time PCR. (D) Protein level of Lats1, YAP1 measured by Western blot. All data are representative of multiple experiments. CTL: control group, MSCcP1P: 1 μM cP1P-primed hdMSC, ns: not significant. *p<0.05, **p<0.01, and ***p<0.001 vs. MSC group.

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  Fig. 4 hdMSCcP1P ameliorates LPS-induced acute lung injury. (A) Histopathological examinations of lung tissue. Mouse lung sections were stained by H&E staining. Original magnification, ×100. Scale bars, 200 μm. (B) Bar graphs showing blinded analysis of lung injury score. (C) Mouse weight before and after treating LPS was measured in each group. (D) Mouse lung weight per body weight was measured in each group. (E) Total protein in BALF was quantified by BCA assay. All data are representative of multiple experiments. CTL: control group, MSCcP1P: 1 μM cP1P-primed hdMSC, ns: not significant. *p<0.05, **p<0.01, and ***p<0.001 vs. LPS groups; #p<0.05 vs. LPS treated MSCcP1P groups.

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  Fig. 5 hdMSCcP1P ameliorates pro-inflammatory cytokine expression and inflammatory signaling pathway of LPS-induced acute lung injury. (A) Macrophages, neutrophils, lymphocytes, and total cells in BALF was counted at each groups. BALF cell was stained by Giemsa staining and counted each group. (B) Pro-inflammatory cytokines in BALF of each group was measured by ELISA. (C) inflammatory signaling factors of each group were quantified by western blot and expression ratio was measured as fold change. All data are representative of multiple experiments. CTL: control group, MSCcP1P: 1 μM cP1P-primed hdMSC, ns: not significant. *p<0.05, **p<0.01, and ***p<0.001 vs. LPS groups; #p<0.05, ##p<0.01 vs. LPS treated MSCcP1P groups.

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  Fig. 6 cP1P acts as a ligand of the S1P receptor of hdMSCs, promoting Hippo signaling. Increased RhoA and reduced Lats1 cause nucleus-translo-cation of YAP/TAZ. YAP/TAZ acts as a transcription factor in TEAD gene site, increasing the expression of CTGF, AREG, and SLUG genes, enhancing the cell migration, invasion, and immune modulation functions of hdMSCs. Enhanced hdMSCs reduce p-STAT1 and iNOS, attenuating inflammation, and recovering damaged tissue LPS-induced ALI mice.

Reference

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