Objective: In sepsis, tumor necrosis factor-alpha (TNF-alpha) contributes to endothelial barrier breakdown. The involvement of Rho A/rho kinase signaling has recently been challenged. Here, we tested the role of cAMP and Rac 1 signaling.
Materials and methods: For this study, we took in vivo measurements of hydraulic conductivity in postcapillary mesenteric venules of adult rats. Measurements of transendothelial electrical resistence (TER), fluorescein isothiocyanate-dextran flux, Western blotting, immunostaining, and enzyme-linked immunosorbent assay-based measurements of cAMP levels and Rho-GTPase activity in human microvascular endothelial cells.
Results: TNF-alpha disrupted endothelial barrier functions in vivo and in vitro. Under these conditions, Rho A activity was significantly increased, whereas Rac 1 activity was decreased and Cdc42 was unaltered. Moreover, cAMP levels were reduced. Rho kinase inhibition, using Y27632, did not prevent TNF-alpha-induced barrier breakdown. In contrast, preincubation with forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 to activate Rac 1 and Rho A abolished TNF-alpha-induced barrier breakdown in vivo and in vitro. Moreover, inactivation of Rac 1 was blocked by F/R-mediated increase of cAMP, whereas Rho A activation was only partially inhibited.
Conclusion: Our data indicate that decrease of cAMP and Rac 1 inactivation, rather than Rho A activation, contribute to TNF-alpha-induced endothelial barrier breakdown in vivo and in vitro.