Role Of HIF-1α In The Regulation Of Vascular Endothelial Hyperpermeability After Severe Burn

The vascular endothelium lining the intima of the blood vessels regulates a variety of functions including host-defense reactions,angiogenesis,and tissue fluid homeostasis. The maintenance of the semi-permeable endothelial barrier is particularly important in controlling the exchange of macromolecules and fluid between the blood and interstitial space.It is well known that the perturbation in the microvascular endothelium play a critical role in the pathogenesis of thermal injury.Changes in tightly connected endothelial cell function and morphology after severe burn can lead to the barrier dysfunction and microvascular leakage,resulting in the development of tissue edema. But the mechanism and the signalling cascades of the barrier dysfunction after severe burn are not fully understood.Transmembrane adherens junctions are the main structures maintaining the barrier function and preventing the transvascular passage of solutes,fluid,and blood cells.On the other hand,contractile forces generated by actomyosin interaction tend to pull the tightly connected cells apart.A disruption of the balance between those adhesive and contractile forces results in endothelial barrier dysfunction and subsequent microvascular leakage.Many agonists,such as histamine and-prostaglandins,can affect the endothelial barrier function,as well as the inflammation cells.Phosphorylation of myosin light chain (MLC) at Ser19/Thr18 by activated myosin light chain kinase(MLCK) plays a critical role in the development and regulation of contractile forces within cells.In addition, activated Rho kinase is known to indirectly result in MLC phosphorylation by inactivating myosin light chain phosphatase(MLCP).The phosphorylated MLC in turn affects the permeability of cultured endothelial monolayers and intact venular endothelium by regulating the interaction of myosin and actin. Changes in endothelial cell function and morphology occur not only at the site of the burn wound but also in the distal internal organs.The burn plasma consisting of oxygen free radicals,proinflammatory mediators,and cytokines plays a particularly important role in the damage of microvascular endothelium.The massive loss of intravascular fluid and proteins results in shock and inadequate perfusion incidents, which plays a critical role in the pathogenesis of multiple organ failure and microvascular endothelium barrier dysfunction after severe burn.HIF-1 is a heterodimer complex consisting of HIF-1α,whose expression is tightly regulated by oxygen concentration,and HIF-1β,which is a constitutively expressed aryl hydrocarbon receptor nuclear translocator.This complex is the master regulator of oxygen homeostasis,and induces a network of genes involved in angiogenesis,erythropoiesis,and glucose metabolism.In addition,FIH,a factor inhibiting the transcription activity of HIF-1αin normoxic conditions,also plays very important role in regulating the oxygen sense and homeostasis.But until now,there is no published data showing that HIF-1αor FIH is the potential regulator of the microvascular endothelium barrier dysfunction.ObjectiveTo investigate the mechanism and the signalling cascades in the regulation of microvascular endothelial hyperpermeability after severe burn,with the focus on the roles of HIF-1αand FIH.Methods1.The Wistar rats inflicted with 30%TBSA full thickness flame burn on the back were killed at Oh,2h,4h,6h,12h,24h postburn,and the vascular permeability and tissue water content of heart,kidney,liver,lung,jejunum and ileum were determined by Evans blue method and by desiccation respectively.The sera taken from the burned rats were applied to the cultured human microvascular endothelial cell(VE) monolayers to test if they can affect the permeability,MLC phosphorylation,cytoskeleton arrangement,and the protein expressions of MLCK,ROCK-Ⅱand ZO-1 in the conditions with or without the specific inhibitors of MLCK or ROCK.2.The permeability,MLC phosphorylation,cytoskeleton arrangement,and the expressions of MLCK and ZO-1 protein were determined in human microvascular endothelial cell monolayers cultured in normoxia(21%O2) or hypoxia(1%O2).It was also tested that if the changes of MLCK protein expression after hypoxia is a transcription or translation-dependent manner.3.The protein expression,nuclear translocation,and DNA binding activity of HIF-1αin human microvascular endothelial cells cultured in normoxia(21%O2) or hypoxia (1%O2) were respectively determined by western blot,immunofluorescence,or electrophoretic mobility shift assay(EMSA).Then,the protein expressions of HIF-1α, HIF-2α,MLCK and the phosphorylated MLC in VE cells stimulated with DMOG under normoxic condition were also assayed by western blot.The plasmid pcDNA3.1-V5-HisA-HIF-1αcontaining the full length of human HIF-1αcDNA was transfected into VE cells to establish the HIF-1αforced expression cell lines,which were then verified by determining the protein and mRNA expression of HIF-1α.In addition,a HIF-1αknock down endothelial cell line was also established by transfection of the plasmid pSUPER-si-HIF-1αwhich contains the small interference RNA targeting to HIF-1α.With the established cell lines,we determined the role of HIF-1αin regulating the permeability and MLC phosphorylation of VE monolayers under hypoxic condition.4.The FIH protein expression was determined in both VE cells stimulated with DMOG in normoxia and VE cells stimulated with hypoxia.A FIH forced expression endothelial cell line was established by transfection of the plasmid pcDNA3-FIH containing the full length of human FIH cDNA,and then verified by determining the protein and mRNA expression of FIH.With the established cell line,we investigated the role of FIH in the regulation of the permeability and MLC phosphorylation in hypoxic VE monolayers.Results1.The vascular permeability and tissue water content of heart,kidney,liver,lung were significantly increased after burn injury.The TER decreased gradually and permeability coefficient increased after the treatment of microvascular endothelial cell monolayers with burned sera.At the same time,the phosphorylated MLC increased,and peaked at 4h after the treatment with burned sera.Furthermore,the cytoskeleton rearrangement and stress fiber formation occurred in VE monolayers treated with burned sera.Pretreatment the cells with ML-9 and Y-27632 effectively attenuated the decrease of TER,the cytoskeleton rearrangement and stress fiber formation induced by burned sera.The burned sera induced increase of phosphorylated MLC was attenuated significantly by addition of ML-9.The results also showed that MLCK,ZO-1 protein were induced markedly in VE cells stimulated with burned sera.2.Compared with those in normoxia,the TER of the human microvascular endothelial cell monolayers decreased gradually and permeability coefficient increased in hypoxic condition.Meanwhile,the phosphorylated MLC increased and peaked at 24h after hypoxia,which was accompanied by the cytoskeleton rearrangement and stress fiber formation.ML-9 pretreatment effectively suppressed the decrease of TER,the cytoskeleton rearrangement,stress fiber formation and the increase of phosphorylated MLC induced by hypoxia.Our results also showed that MLCK,ZO-1 protein were induced markedly by hypoxia.Both actinomycin D and cycloheximide effectively inhibited the increase of MLCK protein and phosphorylated MLC induced by hypoxia.3.Hypoxia induced the accumulation of HIF-1αand HIF-2αin a time-dependent manner.Hypoxia also induced the nuclear translocation,DNA binding,and transcription activity of HIF-1α.DMOG,a hypoxia mimic agent,not only induced the accumulation of HIF-1αand HIF-2αproteins,but also induced the increases of MLCK,phosphorylated MLC,and ZO-1 protein in normoxia.The protein and mRNA levels of HIF-1αwere significantly increased in the established HIF-1αforced expression endothelial cell line. Transfection of small interference RNA targeting HIF-1αeffectively decreased the protein and mRNA levels of HIF-1α,and attenuated the nuclear translocation of HIF-1α. Forced expression of HIF-1αworsened the TER decrease of the monolayers,and augmented the increases of MLCK,phosphorylated MLC,and ZO-1 protein in hypoxia condition.However,knock down of HIF-1αeffectively attenuated the TER decrease of the monolayers and the increased MLCK,phosphorylated MLC,and ZO-1 proteins, which was accompanied by the effective attenuation of the cytoskeleton rearrangement and stress fiber formation.4.Both hypoxia and DMOG treatment decreased the FIH protein.The protein and mRNA levels of FIH were significantly increased in the established FIH forced expression endothelial cell line.Forced expression of FIH markedly attenuated the TER decrease of the monolayers and increased phosphorylated MLC and ZO-1 proteins in cells treated with hypoxia.Knock down of FIH also effectively decreased the cytoskeleton rearrangement and stress fiber formation induced by hypoxia.Conclusions1.The vascular permeability and tissue water content of heart,kidney,liver,lung increase significantly following severe burn injury.The burned sera impair the barrier function of microvascular endothelial cells in vitro by inducing MLC phosphorylation. MLCK protein plays very important role in the regulation of MLC phosphorylation in endothelial cells after burn injury.2.Hypoxia impairs the barrier function of microvascular endothelial cells in vitro, which is mediated by the phosphorylated MLC.The increase of MLCK protein induced by hypoxia is transcriptionally dependent,which in turn regulates the MLC phosphorylation in endothelial cells.Hypoxia also contributes to the regulation of barrier dysfunction by regulating the expression of ZO-1.3.HIF-1αinduced by hypoxia plays very important role in the regulation of microvascular endothelial permeability.HIF-1αinduced the increase of MLCK protein level and the subsequent MLC phosphorylation in hypoxia.HIF-1αalso takes part in the regulation of barrier dysfunction by regulating the expression of ZO-1 protein.4.FIH plays important role in maintaining and regulating the barrier function of microvascular endothelial cells by inhibiting MLC phosphorylation and regulating the expression of ZO-1 protein.