The Role And Mechanism Of Cytoskeleton Rearrangement And Its Relationship With VEGF Or TNF-αon The Hyper-permeability Of Vascularendothelial Cells Induced By Hantavirus Infection

Hantaviruses infect human endothelial cells and are known to causehemorrhagic fever with renal syndrome (HFRS) and Hantavirus pulmonarysyndrome (HPS). The main pathological and clinical characteristics of these twohighly lethal diseasess are enhanced vascular permeability, leading tohemorrhage or acute pulmonary edema in patients days to weeks after the illnessonset. The mechanism of hyper-permeability of vascular endothelial cellsinduced by hantaviruse infection has not been defined. Some studies haveindicated that vascular barrier functions are affected both directly by the virus(for example: the dysfunction of the β3integrin induced by hantavirus infection)and indirectly through the increased synthesis and release of proinflammatorycytokines，such as VEGF (vascular endothelial growth factor) and TNF-α.Endothelial cells (ECs) are targeted by hantaviruses which line the innersurface of blood vessels and form a continuous monolayer as a barrier betweenblood and interstitial compartments. The cytoskeletal reorganization orrearrangement is an important mechanism underlying hyper-permeability, which was considered to be characteristic of the activated contractility. It has been welldocumented that paracellular permeability is controlled by the stability ofintracellular forces and intercellular forces. The former mainly rely on theadhesion complexes providing cell-cell and cell-substrate attachment, which areanchored to the underlying cortical actin ring to membrane and are modulatedby the actin and membrane-binding proteins. The later is maintained by theactomyosin cytoskeleton contraction. The every dynamic process of actinpolymerization allows actin structures rapid reorganization in response toagonist stimulation from the quiescent phenotype, characterized by thick corticalactin ring and the absence of stress fibers, to the activated phenotypecharacterized by thin or no cortical actin and abundant stress fibers andcentrifugal forces Increased. The myosin light chain (MLC) phosphorylation(p-MLC) is one of several key events trigger this reorganization and isconsidered to be characteristic for the state of activated contractility. Thepermeability is associated with opening paracellular gaps, disrupting endothelialjunctions and focal adhesion complexes.Because of the importance of cytoskeleton rearrangement in regulatingendothelial permeability and cytoskeleton rearrangement and it was adjusted byboth β3integrin and VEGF、TNF-α, our study was focus on the the role andmechanism of cytoskeleton rearrangement and its relationship withhyperpereability of vascular endothelial cells that could induced by HTNVinfection and affected by VEGF and TNF-α. These work were laid a basis fordemonstrating the mechanism of hyperpermeability in HFRS patients and fordesigning drugs against the hyperpermeability.Main material and methods1. To test the pathopoiesis effect of hantavirus infection on vascular endothelialpermeability, we test the Trans Epithelial Electrical Resistance (TEER) ofHUVECs in culture infected by HTNV(Hantaan Virus)76-118on various days, because there was not effective animal model.2. To observe the effect and mechanism of cytoskeletal reorganization inHTNV infected HUVECs and affection after treatment with VEGF, we firstlyassessed the function of the β3integrin-VEGFR2complex in HTNV-infectedHUVECs upon treatment with additional VEGF by performing adhesion andmigration assays and a flow cytometry analysis. Then, we test the cytoskeletonrearrangement by immunofluorescence technique; the changes of the importantmolecules RhoA/Rock/p-MLC of the signaling pathway in cytoskeletonrearrangement; and the effect of Rock inhibitor on permeability. The study wasbased on the hypothesis that HTNV binds to the bent, inactivated β3integrinconformers and disrupts β3integrin’s function upon interaction with VEGFR2.Our results supported the previous research, which demonstrated thatHTNV infection inhibits the function of β3integrin in mediating endothelial celladhesion and migration on vitronectin at3to5d post-infection; It also up-regulates the expression of β3integrin and VEGFR2(with a positivecorrelation). Furthermore, we observed that HTNV infection reduced the effectof VEGF on adhesion, migration and the up-regulation of β3integrin expression.The dysfunction and reduced expression of β3integrin may contribute to thereduced effects of VEGF on adhesion and migration. All of the results indicatedthat HTNV infection not only inhibits the function of β3integrin, but alsoinhibits the function of β3integrin-VEGFR2complex. The β3integrin-VEGFR2complex dysfunction might contribute to the effect of VEGF on permeability ofHUVECs induced by HTNV infection.One of the signaling events mediated by the β3integrin-VEGFR2complexis cytoskeletal reorganization. The evidence presented in this study showed thatthe dysregulation of the β3integrin-VEGFR2complex is followed bycytoskeletal reorganization with increasing stress fiber formation and focaladhesion assembly, which provide an anchor for the stress filber on themembrane. We also found that the expressiones of RhoA/Rock/p-MLC were increasedaccompanied with the cytoskeleton rearrangement and hyperpermeability. Andthe Rock inhibition Y27632could alleviate these effect.3. To observe the effect and mechanism of cytoskeletal reorganization in HTNVinfected HUVECs on treatment of TNF-α, we assess the permeability of HTNVinfected HUVECs on treatment of TNF-α, test the cytoskeleton rearrangement,observe the changes of the important molecules RhoA/Rock/p-MLC of thesignaling path in cytoskeleton rearrangement, and measure the effect of Y27632on permeability use the same methods like part2.Main results1. HTNV infection alone could increas the permeability of cultured HUVECs.2. VEGF could increase the permeability of the cultured HUVECs induced byHTNV infection, accompanied with the β3integrin-VEGFR2complexdysfunction and the cytoskeletal rearrangement.3. The expressiones of RhoA/Rock/p-MLC were upregulated and VEGF couldpromote the cytoskeleton rearrangement and hyperpermeability. The Rockinhibitior Y27632could alleviate VEGF effect of hyper-permeability.4. TNF-α could increase the permeability of the cultured HUVECs inducedby HTNV infection accompanied with the cytoskeletal rearrangement.5. The expressiones of RhoA/Rock/p-MLC were increased accompanied withthe effects of TNF-αon cytoskeleton rearrangement and hyperpermeability.And the Rock inhibitor Y27632could alleviate TNF-α effection onpermeability.ConclusionsThe cytoskeleton rearrangement might play an important role on the hyperpermeability of cultured HUVECs induced by HTNV infectrion andenhanced by VEGF or TNF-α.RhoA/Rock/p-MLC might involved in the cytoskeleton rearrangement andRock inhibitor Y27632could alleviate the hyperpermeability of vascularendothelial cells exerted by VEGF and TNF-α.