Effect Of Sphingosine 1-phosphate On Hyper-permeability Response In Scalding Injury

Objective:Increase of vascular permeability is the most important pathological events that play a crucial role in the pathogenesis and outcome during the development of burn injury.Although many advances have been made in reducing the high mortality associated with this injury,there are numerous unresolved questions concerning the nature of burn induced vascular endothelial hyper-permeability.S1P produced by phosphorylation of sphingosine is an abundant lipid mediator in plasma,and serves as a barrier stabilizer via actin organization and intercellular and cell-matrix adherence strengthens.The purpose of this study was to observe the effects of S1P on morphological and functional alteration in cultured endothelial cells(ECs) stimulated by burn plasma and to evaluate the role of exogenous S1P on hyper-permeability response in intact vessels after burn injury.Methods:(1) BABL/c mice,weighing 18-20 g or Wister rat weighing 160-200 g,were randomly divided into 6 groups:sham-burn,sham-burn plus S1P,sham-burn plus DMS,burn,S1P plus burn,burn plus S1P group.(2) Intravital microscopic study of microvascular permeability on mice.Third-order postcapillary venules(V₃,10-50μm) of mesentery were selected for measurement.With the aid of an optical stage of Nikon microscope and digital video-image processing,histograms of fluorescence light intensity distribution in selected fields was measured at various times for construction of a time-dependent integrated optical intensity(IOI) profile,where gray scale values were measured both within(Ii) and outside(Io) the venule,and vascular albumin leakage was determined from the ratio of the interstitium to vascular fluorescence and normalized to background.The increase of venular permeability(Pa) was expressed by relative FITC intensity.(3) Measurement of isolated and perfused venular peameability.A venule of 60-80μm in diameter was dissected from stripped abdomen skin and cannulated with a micropipette on each end.The permeability of the vessel was quantified by measuring the ratio of transvascular flux to transmural concentration difference of albumin.The apparent solute permeability coefficient of albumin(Pa) was calculated to reflect the vascular permeability.(4) Staining of F-actin in endothelial cell of mesentery venule by confocal microscopy.(5) Double staining of F-actin and VE-cadherin imaged by confocal microscopy.Result:Partâ… (1) Mean arterial blood pressure in burned mice slightly lowered than that of controlled group without statistical significance and remained relatively constant throughout the 6h period.(2) Exogenous S1P has no significant effects on vascular permeability in sham-burn animals,the inhibition of S1P synthesis by sphingosine kinase inhibitor DMS could mimic the hyper-permeability response of burn injury,with a Pa ratio of 0.8160±0.0713(p＜0.05). (3) A significant increase in FITC-albumin extravasation in post-capillary venules after burn injury.Pa ratio maintained in remarkably higher in burn group(Pa ratio was 0.7358±0.0654 at 25 min and 0.9141±0.0691 at 50 min) than in sham-burn group(p＜0.05).(4) Pre-burn exogenous applications of S1P attenuated the leakage of albumin within 20 min to 50 min after burn,Pa value were significantly lower compared with burn group(p＜0.05).But post-burn exogenous applications of S1P had no significant effects on vascular permeability in burn animal.Partâ…¡(1) Stimulation of skin venules with 3 h post-burn plasma induced a significant elevation of the apparent permeability coefficient of albumin(Pa),Pa value were significantly higher compared with burn group(p＜0.05).Pre-burn exogenous applications of S1P or post-burn exogenous applications of S1P could both attenuated the increasing Pa after burn injury.Exogenous S1P had no effect in basal permeability,but the inhibition of S1P synthesis by DMS induced a higher Pa,than that of sham-burn group(p＜0.05).(2) In sham-burn mice mesenteric venule,F-actin appeared to be peripheral fibers at outer area of cell near the cell-cell junctions,formed dense fiber bundle called peripheral actin rim(PAR),and the staining line was clear and integrated which showed the grid structure of endothelia cells.S1P could smooth the staining line,but burn injury induced the depolymerization of PAR and disappearance of the grid structure.The inhibition of S1P synthesis by DMS induced almost the same morphological alteration of F-actin as that of burn mice.Pre-burn exogenous applications of S1P attenuated the destruction of peripheral actin rim(PAR) at the outer area of endothelial cells,but post-burn exogenous applications of S1P could not reverse the change after burn injury. Partâ…¢(1) Dose course of morphological changes of F-actin and VE-cadherin induced by exogenous S1P.S1P could stabilize the adherens junction and PAR,smooth the intercellular lining,and such an effect exhibited a dose-dependent enhancement in a range between 0.5-1.0μmol/L.But 10μmol/L S1P stimulation could induce local discontinuities and stress fiber formation.(2) Time course of morphological changes of F-actin and VE-cadherin induced by burn serum.Smooth continuous staining for both F-actin and VE-cadherin along with the intercellular borders of adjacent endothelial cells were seen in normal untreated cells..When endothelial cells were exposed to burn serum,F-actin and VE-cadherin displayed local discontinuities and local stress fiber formation.The changes became more obvious by 6 h burn-serum exposure in F-actin organization, whereby the cells showed an increase and thicking of actin bundles and the formation of stress fibers.VE-cadherin label was seen to consist of numerous saw tooth-shaped structures.(3) The intervention of cultured HUVEC cells with burn serum could cause a noticeable alteration of VE-cadherin spreading at cellular border,displaying a blurred distribution in the location of per se sharp lining without burn serum stimulation.This change was accompanied by the polymerization of F-actin and the formation of stress fiber,resulting in the appearance of intercellular gaps.The addition of S1P before burn plasma stimulation could prevent the obscuration of VE-cadherin and the destruction of peripheral actin rim(PAR) at the outer area of endothelial cells,leading to the preservation of intercellular junctions.Suitable concentration of S1P could stabilize the adherens junction and PAR,smoothing the intercellular lining.The inhibition of S1P synthesis by DMS induced almost the same morphological alteration of VE-cadherin and F-actin in cultured HUVEC as that of burn treated cells.Conclusion.Partâ… S1P might be essential in maintaining the basal vascular barrier function;Burn injury could induce venular hyper-permeability;S1P could be protective in burn insult by preventing the increase of venular permeability.Partâ…¡S1P might be essential in maintaining the rightful distribution of F-actin in microvascular endothelial cell;Burn injury induced hyper-permeability of isolated venular and the depolymerization of PAR,but S1P could prevent or reverse the changes.Partâ…¢S1P could induce F-actin and VE-cadherin distribution in cell peripheral area and such an effect exhibited a dose-dependent enhancement in a range between 0.5～1.0μmol/L.But 10μmol/L S1P stimulation could induce F-actin and VE-cadherin displayed local discontinuities and stress fiber formation;Burn injury could induce F-actin and VE-cadherin redistribution or internalization,which was in time-dependent manner between 30 min～6 h;S1P could prevent and reverse the redistribution of F-action and internalization of VE-cadherin after burn serum stimulation.