HSP27 Phosphorylation Protects Against Endothelial Barrier Dysfunction Under Burn Serum Challenge

Microvascular barrier dysfunction imposes a life-threatening problem to severe burn patients owing to the pathophysiological consequences of plasma loss, organ edema and even shock. Endothelial contraction plays a crucial role in this burn-induced microvascular dysfunction. It leads to opening of the inter-endothelial junctions, allowing a continuous plasma leakage in burn victims. Understanding the mechanisms of endothelial contraction is therefore essential for the prevention of burn-related complications.F-actin rearrangement is an early event in burn-induced endothelial barrier dysfunction.HSP27, a target of p38 MAPK/ MK2 pathway, plays an important role in actin dynamics through phosphorylation.The question of whether HSP27 participates in burn-related endothelial barrier dysfunction has not been identified yet. Here, we showed that burn serum induced a temporal appearance of central F-actin stress fibers followed by a formation of irregular dense peripheral F-actin in pulmonary endothelial monolayer.The appearance of F-actin stress fibers and transient increase of HSP27 phosphorylation occurred prior to the burn serum-induced endothelial hyperpermeability. Overexpressing phospho-mimicking HSP27(HSP27(Asp)) reversed the burn serum-induced peripheral F-actin rearrangement with the augmentation of central F-actin stress fibers, and more importantly, attenuated the burn serum-induced endothelial hyperpermeability; such effects were not observed by HSP27(Ala), a non-phosphorylated mutant of HSP27. HSP27(Asp)overexpression also rendered the monolayer more resistant to barrier disruption caused by Cytochalasin D, a chemical reagent that depolymerizes F-actin specifically. Further study showed that both PP2A-mediated HSP27 dephosphorylation and sumoylation-inhibited MK2 activity contributed to the blunting of HSP27 phosphorylation during the burn serum-induced endothelial hyperpermeability. Our study identifies HSP27 phosphorylation as a protective response against burn serum-induced endothelial barrier dysfunction, and suggests that targeting HSP27 wound be a promising therapeutic strategy in ameliorating burn-induced lung edema and shock development.Objectives:To study the role and mechanism of HSP27 phosphorylation in endothelial barrier dysfuntion under burn serum challenge.Contents:1. To determine the relationship between phosphorylation of HSP27, formation anddistribution of F-actin stress fiber and endothelial monolayer permeability.2. To determine the role of HSP27 phosphorylation in endothelial permeability under burnserum challenge.3. To determine the involvement of PP2 A and MK2 sumoylation in the regulation ofHSP27 phosphorylation in endothelial cells under burn serum challenge.Methods:1. Collection of burn serum from burn patients with second and third-degree burn over50% of their body surface area.2. Culture of rat pulmonary microvascular endothelial cells in transwell monolayer culturesystem and assessment of endothelial monolayer permeability by transendothelialelectrical resistance using Millicell ERS-2 equipment.3. Observation of ZO-1 expression and F-actin distribution in monolayers byimmunofluorescence staining, analysis of cell surface area and intracellular gap area byImage J software.4. Examination of p38 MAPK, MK2 and HSP27 phosphorylation as well as the SUMOmodification of MK2 in endothelial cells under burn serum challenge by Western blot.5. Contruction of adenovirus vector carring the mutant HSP27 gene(phosphorylated andnon-phosphorylated HSP27 gene) or the wild type HSP27 gene, and delivering theminto endothelial cells successfully.Results:1. Burn serum induced endothelial barrier dysfunction in a time depedent manner, asreflected by the decrease of trans-endothelial electrical resistance and increase of theintercellular gaps, concomitant with the decreased cell surface area and the tightjunction disruption.2. Exposure to burn serum resulted in a temporal appearance of central F-actin stressfibers followed by a formation of irregular dense peripheral F-actin in pulmonaryendothelial monolayer.3. While p38 MAPK or MK2 was persistently activated, the phosphorylation of HSP27was only transiently increased prior to the occurrence of endothelial barrierdysfunction.4. HSP27 phosphorylation protects against endothelial barrier dysfunction in associationwith the promotion of F-actin stress fibers, indicating that the transient increase ofp-HSP27 as well as the temporal formation of central F-actin stress fibers in burnserum-treated monolayers may act as an adaptive and protective response against theburn serum-induced endothelial barrier dysfunction.5. Both PP2 A and sumoylation-inhibited MK2 activity contributed to the blunting ofHSP27 phosphorylation during the burn serum-induced endothelial hyperpermeability.6. Our study identified HSP27 phosphorylation as a protective response against burnserum-induced endothelial barrier dysfunction, and suggests that targeting HSP27wound be a promising therapeutic strategy in ameliorating burn-induced lung edemaand shock development.