Endothelial cells and microvascular permeability

It has been shown that nitric oxide attached to albumin behaves as an active nitric oxide compound with an extended half-life. The hallmark of nitrated albumin presence in different vascular beds is the opening of interendothelial junctions. The work describing the cellular pathways involved in opening of endothelial barrier did not investigate the molecular mechanisms behind this effect. We are addressing this issue by investigating the behavior of cellular cytoskeleton when endothelial cell monolayers are exposed to different concentration of NO donors [Sodium Nitroprusside (SNP)] and [Diethylenetriamine/nitric oxide adduct (DETA)] and also to nitrated albumin. Since nitrated albumin may, promote actin modification and thereby may influence junctional integrity and functionality, in the present study we are investigating the molecular mechanisms behind this response. When endothelial cells monolayers were exposed for different time points to excess nitric oxide we observed a redistribution of actin stress fibers inside the cells. Within 5 min, the central stress fibers started to disappear, while the peripheral dense band of actin seemed unaffected. The same pattern was observed after increased time of exposure (60 min), as well as after different concentrations of nitric oxide donors and nitrated albumin. The process is reversible; the central stress fibers reappear when the nitric oxide donors are withdrawn from the media. When endothelial cell monolayers, treated in a similar manner, were analyzed biochemically for their content in globular (G) and fibrillar (F) actin, we found a shift from F to G actin in all conditions tested. This work provides evidence that supports a post-translational modification of cellular actin pool (ADP-ribosylation) induced by the excess of nitric oxide as the principal mechanism involved in the process of opening of the interendothelial junctions. Nitric oxide, through ADP-ribosylation of actin, shifts the dynamic equilibrium that exists between the two pools of actin in favor of G-actin fraction. This shift precipitates a change in the cytoskeletal architecture involved in maintaining cellular shape, and by redistributing some of the main components of inter endothelial junctional complexes disturbs their integrity, which results in vascular barrier opening.