FIG. 5.

(A–L) Micrographs showing localization of VE-cadherin (green) in HUVEC monolayers without (A, D, G) or with added PKH26-labeled WJ-MSC (red) at 2, 16, and 22 h (B, C, E, F, H–L). (A, D, G) Control HUVEC monolayers show presence of VE-cadherin at cell–cell boundaries, with a predominant continuous staining pattern at all time points. (B) Image of coculture with FITC filter only showing VE-cadherin localization in HUVEC after 2 h of coculture with WJ-MSC. Cell–cell junctions with discontinuous VE-cadherin pattern or loss of staining (arrow) can be seen. (C) Superimposed images (FITC/TRITC filters) of the same area showing position of WJ-MSC (red) to the discontinuous cell–cell junctions. They can be seen overlying or in close association with the disrupted cell–cell borders. (E, F) Similar dual images at 16 h of coculture show colocalization of WJ-MSC at disrupted cell–cell junctions. (H, I) At 22 h of coculture, increased numbers of cell–cell borders contain a continuous VE-cadherin staining. In the merged image (I) WJ-MSC can be seen underlying the HUVEC monolayer. Bar=40 μm for images (A–I). (J–L) Merged images at higher magnification at 22 h coculture from different experimental repeats, Bar=20 μm. Full junctional occupancy of VE-cadherin can be seen in cell–cell boundaries overlying WJ-MSC (J). Sub-endothelial position of WJ-MSC can be seen in (K) and (L). Again overlying junctions show full VE-cadherin occupancy. Note discreet shed exosomes can be seen in coculture images. (M) Histogram of percentage of continuous VE-cadherin junctions at different time points for control and cocultured cells. Two-way ANOVA revealed a significant decrease at 2 h (P<0.0001) and significant increase at 22 h (P<0.001) in the cocultures compared with control. Junctional continuity was also statistically significant at each duration of coculture (P<0.0001) (N). Representative western blot showing expression of VE-cadherin. PKH26-labeled WJ-MSC show negative expression (S0h, S2h, S22h) for each duration, HUVEC without WJ-MSC (E0h, E2h, E22h) show no change in expression with duration of culture whereas an upregulation of VE-cadherin can be seen in at 22 h in HUVEC cocultured with WJ-MSC (C0h, C2h, C22h). GAPDH acted as loading control. (O) Histogram showing mean optical density+SEM of normalized VE-cadherin expression from three different blots of control HUVEC and cocultures at 2 and 22 h. No significant changes were seen in the control groups. There was a significant upregulation of VE-cadherin expression at 22 h in the coculture group (P<0.01). Color images available online at www.liebertpub.com/scd