Endothelial barrier dysfunction caused by LPS correlates with phosphorylation of HSP27 in vivo.

Lung edema during sepsis is triggered by formation of gaps between endothelial cells followed by macrophage infiltration. Endothelial gap formation has been proposed to involve changes in the structure of the actin filament cytoskeleton. Heat shock protein 27 (HSP27) is believed to modulate actin filament dynamics or structure, in a manner dependent on its phosphorylation status. We hypothesized that HSP27 may play a role in endothelial gap formation, by affecting actin dependent events in endothelial cells. As there has been no report concerning HSP27 in lung edema in vivo, we examined induction and phosphorylation of HSP27 in lung following LPS injection, as a model of sepsis. In lung, HSP27 mainly localized in capillary endothelial cells of the alveolus, and in smooth muscle cells of pulmonary arteries. HSP27 became significantly more phosphorylated at 3 h after LPS treatment, while the distribution of HSP27 remained unchanged. Pre-treatment with anti-TNFalpha antibody, which has been shown to reduce lung injury, blocked increases in HSP27 phosphorylation at 3 h. HSP27 phosphorylation was also increased in cultured rat pulmonary arterial endothelial cells (RPAEC) by treatment with TNFalpha, LPS, or H2O2. This phosphorylation was blocked by pre-treatment with SB203580, an inhibitor of the upstream kinase, p38 MAP kinase. Increased endothelial permeability caused by H2O2 in vitro was also blocked by SB203580. The amount of actin associated with HSP27 was reduced after treatment with LPS, or H2O2. In summary, HSP27 phosphorylation temporally correlated with LPS induced pathological endothelial cell gap formation in vivo and in a cell culture model system. This is the first report of increased HSP27 phosphorylation associated with pathological lung injury in an animal model of sepsis.