Fig. 4

MSC MV restored the rearrangement of the cytoskeleton protein F-action, the loss of tight junction protein ZO-1, and adherens junction protein VE-cadherin and β-catenin of LPS injured HLMVEC independent of S1P receptor-1. HLMVEC were seeded on cell slides and stained with A phalloidin (green) for F-actin, B Goat anti-rabbit IgG H&L (Alexa flour®488) (green) for ZO-1, and C Goat anti-mouse IgG H&L (Alexa flour®488) (green) for VE-cadherin. F-actin staining in HLMVEC of the control group showed a typical peripheral distribution, and VE-cadherin and ZO-1 staining were strong at the junctions between cells. After being exposed to LPS for 24 h, F-actin of cells reorganized into the center of the cell to form “actin stress fibers,” and the staining of cell adhesion junction protein VE-cadherin and cell tight junction protein ZO-1 was lost, resulting in an increase in cell pore size, which may be the reason for the increased protein permeability. The administration of MSC MV largely prevented the reorganization of cytoskeleton protein F-actin into “actin stress fibers” and restored the staining of VE-cadherin and ZO-1 between HLMVEC cells damaged by LPS. Images are representative for each condition run in triplicates. DAPI (blue) was used to stain the cell nuclei. D Western blot analyses showed that the loss in VE-cadherin total protein levels with LPS injury was partially restored by MSC MV treatment. E Western blot analyses showed that the loss in ZO-1 total protein levels with LPS injury was partially restored by MSC MV treatment. F Western blot analyses showed that the loss in β-catenin total protein levels with LPS injury was also partially restored by MSC MV treatment. The therapeutic effect of MSC MVs on the cytoskeleton structure and cell junction proteins was not abolished by adding S1P receptor-1 antagonist W123. Data are presented as mean ± SD, N = 3, *p < 0.05 versus control using ANOVA with post hoc Tukey HSD test. Abbreviations: MSC, mesenchymal stem cell; MV, microvesicles; LPS, lipopolysaccharide; HLMVEC, human lung microvascular endothelial cells; S1P, sphingosine-1-phosphate; and ANOVA, analysis of variance