Fig 5. Changes in adducin localization parallel to endothelial barrier dysfunction induced by inflammatory mediators.

Barrier integrity of HDMEC control monolayers and monolayers exposed to different inflammatory mediators such as thrombin, LPS and TNFα was evaluated by TER measurements and immunofluorescence analysis. (A) TER in control cells or HDMEC treated with inflammatory agents. Arrows denote the application of mediators. Results are expressed as a mean ± SEM (n = 2). Mediator-induced decrease in TER was accompanied by alterations in the immunostaining pattern not only of the adhesion junctional protein VE-cadherin but also of α-adducin and α-adducin (pSer481). (B-E) HDMEC monolayers exposed to inflammatory mediators or control cells were immunostained for VE-cadherin, α-adducin and α-adducin (pSer481). Staining for F-actin and for nuclei was also accomplished with Alexa Fluor 488 and DAPI, respectively. In comparison to control condition (B) where VE-cadherin was linearly distributed along cell borders and α-adducin as well as α-adducin (pSer481) were present at cell-cell contacts (arrows, B), treatment with LPS (C), thrombin (D) and TNFα (E) led to considerable intercellular gap formation (arrowheads, C-E), paralleled by pronounced increased stress fiber formation and fragmented VE-cadherin distribution. This effect was associated with almost complete loss of α-adducin membrane staining. No differences in the nuclear morphology were observed under these conditions. Scale bar = 20 μm.