LPS increased protein permeability across HLMVEC
A transwell coculture system as we previously described was used to study the effects of MSC MV on protein permeability across HLMVEC monolayer injured by LPS at a concentration of 500 ng/ml (Fig. 2A). FITC-dextran is used as a substitute for albumin to measure the protein permeability of HLMVEC from the upper compartment to the lower compartment and is applied to the upper compartment. As Fig. 2B showed, HLMVEC monolayer damaged by LPS significantly increases the permeability of FITC-dextran through the cell monolayer. The permeability in the LPS group was almost three times higher than in the control group.
MSC MVs restored protein permeability across HLMVEC injured by LPS independent of S1P receptor-1
Early studies have showed that MSC MV could restore the HLMVEC monolayer permeability insulted by cytomix or LPS [23–25]. In addition, we found that MSC MV significantly increased the expression level of sphingosine 1 phosphokinase 1 (SPHK1) mRNA, indicating that S1P signaling may play a role in the restoration of endothelial permeability by MSC MV. However, the underlining mechanism remains unclear. To explore its role in MSC MV treatment for protein permeability across HLMVEC monolayer injured by LPS, we first blocked the S1P receptor-1 of which S1P activation can enhances barrier integrity [26]
HLMVEC monolayer was exposed to 500 ng/ml LPS with or without MSC MV. We used W123 to block the S1P receptor-1 on HLMVEC. After 24 hours, the fluorescence intensity of FITC-dextran 70 kDa in the upper chamber and lower chamber was detected by the plate reader. As Fig. 3 showed, exposure to LPS 500 ng/ml further increased protein permeability across HLMVEC to 317% of control. However, when MSC MV (60ul) were added to the top chamber of the transwell coculture system, the protein permeability was significantly decreased to 154% of control. The same treatment effect of MSC MV was observed when adding W123 (185% of control). No significant difference in protein permeability was showed between the LPS + MV and LPS + MV + W123 groups, indicating that the treatment effect of MSC MV on protein permeability across HLMVEC injured by LPS was independent of S1P receptors on HLMVEC.
MSC MV restored cytoskeleton protein F-action, tight junction protein ZO-1 and adherens junction protein VE-cadherin and β-catenin of LPS injured HLMVEC via independent of S1P receptor-1
To better understand the role of S1P in MSC MV treatment for endothelial permeability, we blocked the S1P receptor on endothelial cells and used immunofluorescence and western blotting to study the distribution of the cytoskeletal protein F-actin, the adhesion junction protein VE-cadherin and β-catenin, and the tight junction protein zonula occludens-1 (ZO-1). The results showed that MSC MV significantly improved the rearrangement of cytoskeletal protein F-actin into "actin stress fibers" in HLMVEC damaged by LPS (Fig. 4A). Moreover, MSC MV treatment significantly prevented the loss of the adhesion-associated protein VE-cadherin and β-catenin (Fig. 4C, D&F), as well as tight junction protein ZO-1 (Fig. 4B&E) in LPS injured HMVEC. The blockage of S1P receptor on HLMVEC had no impact on the effect of MSC MV in F-action, ZO-1, and adherens junction protein VE-cadherin and β-catenin, further demonstrated that the therapeutic effect of MSC MV on protein permeability was not dependent on S1P receptor.
MSC MV increased SPHK1 and intracellular S1P levels in LPS injured HLMVEC
Due to the presence of mRNA, microRNA proteins/peptides, lipids and organelles, MSC MV has been shown to be biologically active. In our previous study, we also found that The expression level of sphingosine 1 phosphokinase 1 (SPHK1) mRNA in damaged HLMVEC increased significantly after MSC MV treatment[23]. To further understand the role of S1P in the therapeutic effects of MSC MVs, we measured SPHK1 and S1P in HLMVEC as well as S1P in HLMVEC supernatant. We found that as compared with LPS group, the expression of SPHK1 protein in HLMVEC in both the LPS + MV group and LPS + MV + W123 groups significantly increased. However, there was no significant difference between the LPS + MV group and LPS + MV + W123 groups (Fig. 5C&D). Moreover, ELISA results showed that as compared with the LPS group, the level of S1P in HLMVEC in both the LPS + MV and LPS + MV + W123 groups increased and the levels of S1P in HLMVEC supernatant decreased statistically. But no significant difference was found in the S1P levels in HLMVEC and in HLMVEC supernatant between the LPS + MV and LPS + MV + W123 groups (Fig. 5A&B). Given that the therapeutic effect of MSC MV on HLMVEC monolayer permeability was not abolished by the blockage of S1P receptor-1 and the generation of S1P in HLMVEC was increased, we could concluded that MSC MV improved protein permeability across LPS injured HLMVEC partly by increasing S1P level in HLMVEC.
MSC MV promoted Ca2+ mobilization and activated Rac1 pathway in LPS injured HLMVEC by increasing its intracellular S1P level.
Rac activity is required for S1P-induced adherens junction assembly and cytoskeleton rearrangement [27]. A previous study found that increasing the level of S1P in the cytoplasm of endothelial cells through photolysis could enhance intracellular Ca2+ mobilization, thereby inducing the activation of Rac1/IQGAP1 pathway, causing the rearrangement of cytoskeletal protein, the adherens junction protein-VE-cadherin, β-catenin, thereby reducing vascular endothelial permeability. The administration of S1P receptor-1 antagonist can block the improvement of vascular permeability caused by exogenous S1P, but has no effect on the improvement of vascular permeability caused by the increase of intracellular S1P level caused by photolysis[28]. Therefore, the accumulation of S1P in cells can increase the intracellular calcium ion mobilization, thereby directly activating the Rac1 pathway and enhancing the endothelial barrier function.
To explore the downstream signaling moleculars of S1P, we used laser confocal to measure the Ca2+ mobilization and Rac activation in HLMVEC. Laser confocal results (Fig. 6) showed that LPS impaired the Ca2+ mobilization in HLMVEC dramatically. MSC MV treatment significantly improved the mobilization of Ca2+ in LPS injured HLMVEC. And this therapeutic effect of MSC MV was not affected by adding S1P receptor-1 atagonist W123. Meanwhlie, as compared with LPS group, Rac1 activation in HLMVEC in both the LPS + MV and LPS + MV + W123 groups significantly increased, but no difference was found in the activation of Rac1 in HLMVEC between the LPS + MV and LPS + MV + W123 groups (Fig. 7). The results implied that MSC MV promoted Ca2+ mobilization and activated Rac1 pathway in LPS injured HLMVEC by increasing intracellular S1P independent of S1P receptor-1.