LPS, Burn Rat PMN And Burn Rat Serum Induced Functional Changes In Endothelial Cytoskeleton And VE-cadherin

ObjectivesDespite recent therapeutic advances, inflammatory conditions such as acute lung injury, acute respiratory distress syndrome, and sepsis continue to result in high rates of patient morbidity and mortality. Centrally involved in the pathogenesis of these processes and now recognized as a cardinal feature of inflammation, increased vascular permeability contributes to the profound pathophysiological derangements observed in these disorders.Endothelium injury is accompanied by altered cell morphology, intercellular gap formation, and increased transendothelial permeability. It was possible that cytoskeletal alterations in LPS-induced endothelium injury were closely linked to intercellular gap formation and endothelial barrier dysfunction. There were few reports on detailed alterations of cytoskeleton in morphological changes of LPS-induced endothelial injury. This study is to investigate dose and time-dependent LPS-induced morphological changes of actin cytoskeleton and VE-cadherin by visualizing their distribution and measuring their contents in ECs. The study also examined LPS-induced [Ca2+], change. In addition, morphological changes of F-actin and G-actin in ECs subjected to burn-activated PMNs and burn rat serum were also detected.Methods:1. To visualize the distribution of actin, HUVECs (ECV-304) subjected to different stimulations were fixed and permeabilized with 2% formaldehydum and 0.5%Triton X-100 solution dissolved in PBS for 20 min at room temperature. Then cells were incubated with rhodamine-phalloidin (2 u/ml) or Oregon Green-Dnase I (0. 6μmol/L) for 40 min to stain F-actin or G-actin, then washed three times with cool PBS. Petri capsule was then mounted to fluorescence microscope, images were recorded on films with Kodak 200. The contents of F-actin and G-actin inECs were measured with flow cytometer technology.2. To visualize the distribution of VE-cadherin, HUVECs (ECV-304) subjected to different stimulations were fixed with 2% formaldehydum solution dissolved in PBS for 20 min, then permeabilized with 0.5%Triton X-100 solution for 10 min at room temperature. Cells were incubated with 2% rabbit serum in PBS for 1h to block nonspecific antibody binding and then incubated with primary VE-cadherin mAb (1:100) , followed by FITC-conjugated secondary antibody (1:200) incubation. Cells were then washed three times with PBS. Petri capsule was then mounted in fluorescence microscope, images were recorded on films with Kodak 200. The expression level of VE-cadherin was detected by fluorescence spectrophotometer represented by relative absorbance.3. For calcium measurements, HUVECs were loaded with calcium specific probe Fura 3-AM. After LPS was added to the buffer at various concentration, [Ca2+], was assessed with confocal microscopy.Data analysisData are reported as mean±SD. ANOVA was used to evaluate the significant of intergroup differences. A value of P<0.05 was considered significant for the comparisons and P<0.01 as very significant.Results1. F-actin was mainly distributed in EC cellular cortex under normal condition, while G-actin was seen in perinulear and nuclear area. HUVEC exposed to LPS showed F-actin disorganization with formation of stress fibers, filopodia, and lamellipodia. A higher concentration of LPS caused the assembly and polymerization of actin filament and finally disruptions of them. G-actin was rearranged from nuclear to cytoplasmic region under LPS stimulations. The marked actin disorganization was accompanied by alteration of F-actin and G-actin pools in EC exposed to LPS while thecontent of F-actin decreased and the level of G-actin elevated, both in dose-and time-dependent patterns.2. Normal VE-cadherin was distributed at cell-to-cell junction with uniform staining and EC maintained tight connection. Treatment of HUVEC with LPS caused serrate shape of VE-cadherin staining and formation of intercellular gaps. LPS reduced VE-cadherin expression represented by relative fluorescence abs...