细胞骨架重构在VEGF与TNF-α促进汉坦病毒感染致血管内皮细胞通透性增高中的作用及其机制
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摘要
汉坦病毒（hantanvirus）是肾综合征出血热（hemorrhagic fever with renalsyndrome, HFRS）和汉坦病毒肺综合征（hantavirus pulmonary syndrome,HPS）的病原体。致病性汉坦病毒感染人体后以毛细血管和小血管的内皮细胞为主要靶细胞，在感染后数周内引发以血管渗漏综合征（vascular leakagesyndrome, CLS）和肾功能障碍为主要临床表现的临床综合征。目前血管内皮细胞损伤的机制研究主要包括病毒与其受体β3整合素结合后直接影响内皮细胞功能致病学说和以血管内皮生长因子（vascular endothelial growthfactor, VEGF）、肿瘤坏死因子（Tumor Necrosis Factor-α, TNF-α）等炎症介质升高和相关免疫细胞功能变化等免疫学致病学说。而具体机制有待深入研究。
细胞骨架重构是调节血管内皮通透性升高的重要机制，而β3整合素和VEGF、TNF-α等均参与细胞骨架的调节。所以本研究以汉滩病毒（hantaanvirus, HTNV，致病性汉坦病毒中的主要型别）和体外培养的人脐静脉内皮细胞（human umbilical vein endothelial cell, HUVECs）为研究对象，以细胞骨架为切入点，研究细胞骨架重构是否参与了汉坦病毒感染以及VEGF、TNF-α对体外培养的血管内皮细胞通透性的影响；观察调节细胞骨架重构的重要分子RhoA/Rock/p-MLC的变化，以及Rock激酶抑制剂Y27632能否阻断或部分阻断血管内皮通透性的变化。旨在探讨汉坦病毒感染所致的血管内皮高通透性的发病机理，并为下一步深入研究减轻细胞高通透性的药物研究提供实验依据。
主要研究方法和内容
1. HTNV感染对HUVECs通透性影响
实验（一）首先扩增并滴定HTNV76-118毒株并感染体外培养的HUVECs。运用跨内皮电阻测定（trans epithelial electrical resistance,TEER）、免疫荧光染色、流式细胞术等技术观察HTNV感染HUVECs不同时间点对内皮细胞通透性的影响，为后续实验提供相关数据及操作经验。
2.细胞骨架重构在VEGF促进HTNV感染致HUVECs高通透性中的作用及机制
实验（二）中以体外培养的HUVECs为对象，应用粘附实验、Transwell技术及流式细胞技术，观察VEGF对HTNV感染HUVECs的通透性、粘附能力和迁移能力的改变，分析β3与VEGFR2表达数量的变化；用荧光染色技术标记显示细胞骨架的重构；电子显微镜观察细胞间连接结构的变化；免疫蛋白印迹（western-blot）、逆转录实时定量PCR（q RT-PCR）方法检测β3-VEGFR2功能复合物下游参与细胞骨架重构的重要分子RhoA/Rock激酶、磷酸化的肌球蛋白轻链（myosin light chain phosphorylation, P-MLC）蛋白和mRNA水平上的变化。以上实验拟证实致病性汉坦病毒感染不仅影响β3整合素的功能，而且影响β3-VEGFR2复合物的功能，这可能是HFRS病程中VEGF水平升高而加剧血管内皮通透性的原因之一；RhoA/Rock/p-MLC分子通路参与的细胞骨架重构可能是VEGF促进HTNV感染HUVECs通透性升高的机制之一；Rock激酶的抑制剂Y27632可以部分阻断或减弱VEGF促进HTNV感染HUVECs高通透性的作用。
3.细胞骨架重构在TNF-α致HTNV感染致HUVECs高通透性中的作用
既往研究显示TNF-α的增高也参与了HFRS病程血管通透性的调节。而细胞骨架重构可能是TNF-α增加内皮细胞的通透性的重要通路。
实验（三）采用内皮细胞电阻仪、免疫荧光染色、western-blot、qRT-PCR、电子显微镜等与第二部分实验中类似的实验方法观察TNF-α对HTNV感染的HUVECs通透性的作用，伴随通透性改变的细胞骨架重构情况、相邻细胞间连接结构的变化，RhoA/Rock/p-MLC等分子表达的变化；以及Y27632对上述作用有否影响。
主要结果：
1.单纯HTNV感染可以导致体外培养的HUVECs通透性升高。
2. VEGF可以明显促进HTNV感染HUVECs通透性的升高，并伴随有β3整合素-VEGFR2复合体功能失调及细胞骨架的重构。
3. RhoA/Rock/P-MLC分子通路可能参与VEGF介导的细胞骨架重构所致的通透性升高；Rock激酶抑制剂Y27632可以明显减轻上述作用。
4. TNF-α可明显促进HTNV感染HUVECs通透性的升高，并伴随有细胞骨架的重构。
5. RhoA/Rock/P-MLC分子通路可能参与TNF-α介导的细胞骨架重构所致的通透性升高；Rock激酶抑制剂Y27632可以明显减轻上述作用。
结论
1.细胞骨架重构可能是VEGF和TNF-α促进HTNV感染致HUVECs的高通透性的重要机制之一。
2. RhoA/Rock/p-MLC分子通路可能是调节上述细胞骨架重构的重要信号途径，Rock激酶抑制剂Y27632具有明显减轻VEGF及TNF-α促进HTNV感染HUVECs高通透性的作用。
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 methods
1. 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 results
1. 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.
Conclusions
The 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.
细胞骨架重构是调节血管内皮通透性升高的重要机制，而β3整合素和VEGF、TNF-α等均参与细胞骨架的调节。所以本研究以汉滩病毒（hantaanvirus, HTNV，致病性汉坦病毒中的主要型别）和体外培养的人脐静脉内皮细胞（human umbilical vein endothelial cell, HUVECs）为研究对象，以细胞骨架为切入点，研究细胞骨架重构是否参与了汉坦病毒感染以及VEGF、TNF-α对体外培养的血管内皮细胞通透性的影响；观察调节细胞骨架重构的重要分子RhoA/Rock/p-MLC的变化，以及Rock激酶抑制剂Y27632能否阻断或部分阻断血管内皮通透性的变化。旨在探讨汉坦病毒感染所致的血管内皮高通透性的发病机理，并为下一步深入研究减轻细胞高通透性的药物研究提供实验依据。
主要研究方法和内容
1. HTNV感染对HUVECs通透性影响
实验（一）首先扩增并滴定HTNV76-118毒株并感染体外培养的HUVECs。运用跨内皮电阻测定（trans epithelial electrical resistance,TEER）、免疫荧光染色、流式细胞术等技术观察HTNV感染HUVECs不同时间点对内皮细胞通透性的影响，为后续实验提供相关数据及操作经验。
2.细胞骨架重构在VEGF促进HTNV感染致HUVECs高通透性中的作用及机制
实验（二）中以体外培养的HUVECs为对象，应用粘附实验、Transwell技术及流式细胞技术，观察VEGF对HTNV感染HUVECs的通透性、粘附能力和迁移能力的改变，分析β3与VEGFR2表达数量的变化；用荧光染色技术标记显示细胞骨架的重构；电子显微镜观察细胞间连接结构的变化；免疫蛋白印迹（western-blot）、逆转录实时定量PCR（q RT-PCR）方法检测β3-VEGFR2功能复合物下游参与细胞骨架重构的重要分子RhoA/Rock激酶、磷酸化的肌球蛋白轻链（myosin light chain phosphorylation, P-MLC）蛋白和mRNA水平上的变化。以上实验拟证实致病性汉坦病毒感染不仅影响β3整合素的功能，而且影响β3-VEGFR2复合物的功能，这可能是HFRS病程中VEGF水平升高而加剧血管内皮通透性的原因之一；RhoA/Rock/p-MLC分子通路参与的细胞骨架重构可能是VEGF促进HTNV感染HUVECs通透性升高的机制之一；Rock激酶的抑制剂Y27632可以部分阻断或减弱VEGF促进HTNV感染HUVECs高通透性的作用。
3.细胞骨架重构在TNF-α致HTNV感染致HUVECs高通透性中的作用
既往研究显示TNF-α的增高也参与了HFRS病程血管通透性的调节。而细胞骨架重构可能是TNF-α增加内皮细胞的通透性的重要通路。
实验（三）采用内皮细胞电阻仪、免疫荧光染色、western-blot、qRT-PCR、电子显微镜等与第二部分实验中类似的实验方法观察TNF-α对HTNV感染的HUVECs通透性的作用，伴随通透性改变的细胞骨架重构情况、相邻细胞间连接结构的变化，RhoA/Rock/p-MLC等分子表达的变化；以及Y27632对上述作用有否影响。
主要结果：
1.单纯HTNV感染可以导致体外培养的HUVECs通透性升高。
2. VEGF可以明显促进HTNV感染HUVECs通透性的升高，并伴随有β3整合素-VEGFR2复合体功能失调及细胞骨架的重构。
3. RhoA/Rock/P-MLC分子通路可能参与VEGF介导的细胞骨架重构所致的通透性升高；Rock激酶抑制剂Y27632可以明显减轻上述作用。
4. TNF-α可明显促进HTNV感染HUVECs通透性的升高，并伴随有细胞骨架的重构。
5. RhoA/Rock/P-MLC分子通路可能参与TNF-α介导的细胞骨架重构所致的通透性升高；Rock激酶抑制剂Y27632可以明显减轻上述作用。
结论
1.细胞骨架重构可能是VEGF和TNF-α促进HTNV感染致HUVECs的高通透性的重要机制之一。
2. RhoA/Rock/p-MLC分子通路可能是调节上述细胞骨架重构的重要信号途径，Rock激酶抑制剂Y27632具有明显减轻VEGF及TNF-α促进HTNV感染HUVECs高通透性的作用。
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 methods
1. 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 results
1. 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.
Conclusions
The 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.
引文
1. Bjorkstrom NK, Lindgren T, Stoltz M, Fauriat C, Braun M, Evander M,Michaelsson J, Malmberg KJ, Klingstrom J, Ahlm C, Ljunggren HG. Rapidexpansion and long-term persistence of elevated NK cell numbers in humansinfected with hantavirus. J Exp Med2011;208:13-21.
2. Raftery MJ, Kraus AA, Ulrich R, Kruger DH, Schonrich G. Hantavirus infection ofdendritic cells. J Virol2002;76:10724-33.
3. Wang M, Wang J, Zhu Y, Xu Z, Yang K, Yang A, Jin B. Cellular immune responseto Hantaan virus nucleocapsid protein in the acute phase of hemorrhagic fever withrenal syndrome: correlation with disease severity. J Infect Dis2009;199:188-95.
4. Urbani S, Amadei B, Fisicaro P, Tola D, Orlandini A, Sacchelli L, Mori C, MissaleG, Ferrari C. Outcome of acute hepatitis C is related to virus-specific CD4functionand maturation of antiviral memory CD8responses. Hepatology2006;44:126-39.
5. Dvorak HF. Discovery of vascular permeability factor (VPF). Exp Cell Res2006;312:522-6.
6. Srikiatkhachorn A, Ajariyakhajorn C, Endy TP, Kalayanarooj S, Libraty DH, GreenS, Ennis FA, Rothman AL. Virus-induced decline in soluble vascular endothelialgrowth receptor2is associated with plasma leakage in dengue hemorrhagic Fever.J Virol2007;81:1592-600.
7. Wallez Y, Huber P. Endothelial adherens and tight junctions in vascularhomeostasis, inflammation and angiogenesis. Biochim Biophys Acta2008;1778:794-809.
8. Niikura M, Maeda A, Ikegami T, Saijo M, Kurane I, Morikawa S. Modification ofendothelial cell functions by Hantaan virus infection: prolonged hyper-permeabilityinduced by TNF-alpha of hantaan virus-infected endothelial cell monolayers. ArchVirol2004;149:1279-92.
9. Oelschlegel R, Kruger DH, Rang A. MxA-independent inhibition of Hantaan virusreplication induced by type I and type II interferon in vitro. Virus Res2007;127:100-5.
10.王平忠，李宜川，陈延平，等.肾综合征出血热患者TNF-α、IL-6、IL-4、 IFN-γ水平变化及意义.西南国防医药2007;17:396-398.
11. Wimer BM. Implications of the analogy between recombinant cytokine toxicitiesand manifestations of hantavirus infections. Cancer Biother Radiopharm1998;13:193-207.
12.王平忠，王九平，王雅格，等.肾综合征出血热患者IL-8、IP-10和RANTES的水平变化及意义.中国现代医学杂志2008;18:1248-1250.
13. Liu JM, Zhu Y, Xu ZW, Ouyang WM, Wang JP, Liu XS, Cao YX, Li Q, Fang L,Zhuang R, Yang AG, Jin BQ. Dynamic changes of apoptosis-inducing ligands andTh1/Th2like subpopulations in Hantaan virus-induced hemorrhagic fever withrenal syndrome. Clin Immunol2006;119:245-51.
14. Mou DL, Wang YP, Huang CX, Li GY, Pan L, Yang WS, Bai XF. Cellular entry ofHantaan virus A9strain: specific interactions with beta3integrins and a novel70kDa protein. Biochem Biophys Res Commun2006;339:611-7.
15.牟丹蕾，白雪帆*，黄长形，等.人整合素αvβ3在CHO细胞表达的研究.中华微生物学和免疫学杂志200323:618-624.
16. Switala-Jelen K, Dabrowska K, Opolski A, Lipinska L, Nowaczyk M, Gorski A.The biological functions of beta3integrins. Folia Biol (Praha)2004;50:143-52.
17. Luo BH, Springer TA. Integrin structures and conformational signaling. Curr OpinCell Biol2006;18:579-86.
18. Mould AP, Travis MA, Barton SJ, Hamilton JA, Askari JA, Craig SE, MacdonaldPR, Kammerer RA, Buckley PA, Humphries MJ. Evidence that monoclonalantibodies directed against the integrin beta subunit plexin/semaphorin/integrindomain stimulate function by inducing receptor extension. J Biol Chem2005;280:4238-46.
19. Raymond T, Gorbunova E, Gavrilovskaya IN, Mackow ER. Pathogenichantaviruses bind plexin-semaphorin-integrin domains present at the apex ofinactive, bent alphavbeta3integrin conformers. Proc Natl Acad Sci U S A2005;102:1163-8.
20. Zhang JL, Wang JL, Gao N, Chen ZT, Tian YP, An J. Up-regulated expression ofbeta3integrin induced by dengue virus serotype2infection associated with virusentry into human dermal microvascular endothelial cells. Biochem Biophys ResCommun2007;356:763-8.
21. Tang QH, Zhang YM, Xu YZ, He L, Dai C, Sun P. Up-regulation of integrin beta3expression in porcine vascular endothelial cells cultured in vitro by classical swinefever virus. Vet Immunol Immunopathol2010;133:237-42.
22. Gahmberg CG FS, Nurmi SM. Regulation of integrin activity and signaling.Biochim Biophysica Acta2009;1790:431-444.
23. Mahabeleshwar GH, Feng W, Phillips DR, Byzova TV. Integrin signaling is criticalfor pathological angiogenesis. J Exp Med2006;203:2495-507.
24. Gahmberg CG, Fagerholm SC, Nurmi SM, Chavakis T, Marchesan S, Gronholm M.Regulation of integrin activity and signalling. Biochim Biophys Acta2009;1790:431-44.
25. Martin KH, Slack JK, Boerner SA, Martin CC, Parsons JT. Integrin connectionsmap: to infinity and beyond. Science2002;296:1652-3.
26. Kumar CC. Signaling by integrin receptors. Oncogene1998;17:1365-73.
27. Yanagihara R, Silverman DJ. Experimental infection of human vascular endothelialcells by pathogenic and nonpathogenic hantaviruses. Arch Virol1990;111:281-6.
28. Gavrilovskaya IN, Shepley M, Shaw R, Ginsberg MH, Mackow ER. beta3Integrins mediate the cellular entry of hantaviruses that cause respiratory failure.Proc Natl Acad Sci U S A1998;95:7074-9.
29. Zaki SR, Greer PW, Coffield LM, Goldsmith CS, Nolte KB, Foucar K, FeddersenRM, Zumwalt RE, Miller GL, Khan AS, et al. Hantavirus pulmonary syndrome.Pathogenesis of an emerging infectious disease. Am J Pathol1995;146:552-79.
30. Gavrilovskaya IN, Peresleni T, Geimonen E, Mackow ER. Pathogenic hantavirusesselectively inhibit beta3integrin directed endothelial cell migration. Arch Virol2002;147:1913-31.
31. Gavrilovskaya IN, Gorbunova EE, Mackow NA, Mackow ER. Hantaviruses directendothelial cell permeability by sensitizing cells to the vascular permeability factorVEGF, while angiopoietin1and sphingosine1-phosphate inhibithantavirus-directed permeability. J Virol2008;82:5797-806.
32. Gavard J, Patel V, Gutkind JS. Angiopoietin-1prevents VEGF-induced endothelialpermeability by sequestering Src through mDia. Dev Cell2008;14:25-36.
33. Lampugnani MG, Dejana E. Adherens junctions in endothelial cells regulate vesselmaintenance and angiogenesis. Thromb Res2007;120Suppl2:S1-6.
34. Mackow ER, Gavrilovskaya IN. Hantavirus regulation of endothelial cell functions.Thromb Haemost2009;102:1030-41.
35. Buranda T, Wu Y, Perez D, Jett SD, BonduHawkins V, Ye C, Edwards B, Hall P,Larson RS, Lopez GP, Sklar LA, Hjelle B. Recognition of decay accelerating factorand alpha(v)beta(3) by inactivated hantaviruses: Toward the development ofhigh-throughput screening flow cytometry assays. Anal Biochem2010;402:151-60.
36. Krautkramer E, Zeier M. Hantavirus causing hemorrhagic fever with renalsyndrome enters from the apical surface and requires decay-accelerating factor(DAF/CD55). J Virol2008;82:4257-64.
37. Choi Y, Kwon YC, Kim SI, Park JM, Lee KH, Ahn BY. A hantavirus causinghemorrhagic fever with renal syndrome requires gC1qR/p32for efficient cellbinding and infection. Virology2008;381:178-83.
38.徐芳玲，杨占秋.汉坦病毒受体.病毒学报2004;20:288-290.
39. Ogino M, Yoshimatsu K, Ebihara H, Arikawa J. N-acetylgalactosamine(GalNAc)-specific lectins mediate enhancement of Hantaan virus infection. ArchVirol1999;144:1765-77.
40. Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability.Physiol Rev2006;86:279-367.
41. Vestweber D, Winderlich M, Cagna G, Nottebaum AF. Cell adhesion dynamics atendothelial junctions: VE-cadherin as a major player. Trends Cell Biol2009;19:8-15.
42. Harris ES NW. VE-cadherin: at the front, center, and sides of endothelial cellorganization and function. Curr Opin Cell Biol2010;22651-8.
43.赵克森，黄巧冰.血管通透性增高的基本机制.中国病理生理杂志2003;19:549-553.
44.李彧，王伟，王平忠.肾综合征出血热外周静脉血细胞因子的变化与病情的关系.中国病毒病杂志2011;1:260-263.
45. Peterson LJ, Rajfur Z, Maddox AS, Freel CD, Chen Y, Edlund M, Otey C, BurridgeK. Simultaneous stretching and contraction of stress fibers in vivo. Mol Biol Cell2004;15:3497-508.
46. Thiagarajan P, Le A, Snuggs MB, VanWinkle B. The role of carboxy-terminalglycosaminoglycan-binding domain of vitronectin in cytoskeletal organization andmigration of endothelial cells. Cell Adhes Commun1996;4:317-25.
47. Lee TY, Gotlieb AI. Microfilaments and microtubules maintain endothelial integrity.Microsc Res Tech2003;60:115-27.
48. Jacobson JR, Garcia JG. Novel therapies for microvascular permeability in sepsis.Curr Drug Targets2007;8:509-14.
49. Bogatcheva NV, Verin AD. The role of cytoskeleton in the regulation of vascularendothelial barrier function. Microvasc Res2008;76:202-7.
50. Bogatcheva NV, Verin AD, Wang P, Birukova AA, Birukov KG, Mirzopoyazova T,Adyshev DM, Chiang ET, Crow MT, Garcia JG. Phorbol esters increase MLCphosphorylation and actin remodeling in bovine lung endothelium withoutincreased contraction. Am J Physiol Lung Cell Mol Physiol2003;285:L415-26.
51. Tiruppathi C, Minshall RD, Paria BC, Vogel SM, Malik AB. Role of Ca2+signaling in the regulation of endothelial permeability. Vascul Pharmacol2002;39:173-85.
52. van Nieuw Amerongen GP, van Hinsbergh VW. Endogenous RhoA inhibitorprotects endothelial barrier. Circ Res2007;101:7-9.
53. Gorovoy M, Neamu R, Niu J, Vogel S, Predescu D, Miyoshi J, Takai Y, Kini V,Mehta D, Malik AB, Voyno-Yasenetskaya T. RhoGDI-1modulation of the activityof monomeric RhoGTPase RhoA regulates endothelial barrier function in mouselungs. Circ Res2007;101:50-8.
54. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature2002;420:629-35.
55. Wojciak-Stothard B, Ridley AJ. Rho GTPases and the regulation of endothelialpermeability. Vascul Pharmacol2002;39:187-99.
56. Ivanov AI, Parkos CA, Nusrat A. Cytoskeletal regulation of epithelial barrierfunction during inflammation. Am J Pathol2010;177:512-24.
57. Amano M, Nakayama M, Kaibuchi K. Rho-kinase/ROCK: A key regulator of thecytoskeleton and cell polarity. Cytoskeleton (Hoboken)2010;67:545-54.
58. Hirano S, Rees RS, Yancy SL, Welsh MJ, Remick DG, Yamada T, Hata J, GilmontRR. Endothelial barrier dysfunction caused by LPS correlates with phosphorylationof HSP27in vivo. Cell Biol Toxicol2004;20:1-14.
59. Tar K, Csortos C, Czikora I, Olah G, Ma SF, Wadgaonkar R, Gergely P, Garcia JG,Verin AD. Role of protein phosphatase2A in the regulation of endothelial cellcytoskeleton structure. J Cell Biochem2006;98:931-53.
60. Borbiev T, Birukova A, Liu F, Nurmukhambetova S, Gerthoffer WT, Garcia JG,Verin AD. p38MAP kinase-dependent regulation of endothelial cell permeability.Am J Physiol Lung Cell Mol Physiol2004;287:L911-8.
61. Bogatcheva NV, Birukova A, Borbiev T, Kolosova I, Liu F, Garcia JG, Verin AD.Caldesmon is a cytoskeletal target for PKC in endothelium. J Cell Biochem2006;99:1593-605.
62. Lee YH, Kayyali US, Sousa AM, Rajan T, Lechleider RJ, Day RM. Transforminggrowth factor-beta1effects on endothelial monolayer permeability involve focaladhesion kinase/Src. Am J Respir Cell Mol Biol2007;37:485-93.
63. Bogatcheva NV, Adyshev D, Mambetsariev B, Moldobaeva N, Verin AD.Involvement of microtubules, p38, and Rho kinases pathway in2-methoxyestradiol-induced lung vascular barrier dysfunction. Am J Physiol LungCell Mol Physiol2007;292:L487-99.
64. Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, Garcia JG.Microtubule disassembly increases endothelial cell barrier dysfunction: role ofMLC phosphorylation. Am J Physiol Lung Cell Mol Physiol2001;281:L565-74.
65. Birukova AA, Adyshev D, Gorshkov B, Bokoch GM, Birukov KG, Verin AD.GEF-H1is involved in agonist-induced human pulmonary endothelial barrierdysfunction. Am J Physiol Lung Cell Mol Physiol2006;290:L540-8.
66. Sehrawat S, Cullere X, Patel S, Italiano J, Jr., Mayadas TN. Role of Epac1, anexchange factor for Rap GTPases, in endothelial microtubule dynamics and barrierfunction. Mol Biol Cell2008;19:1261-70.
67. Gonzalez-Mariscal L, Tapia R, Chamorro D. Crosstalk of tight junctioncomponents with signaling pathways. Biochim Biophys Acta2008;1778:729-56.
68. Harhaj NS, Antonetti DA. Regulation of tight junctions and loss of barrier functionin pathophysiology. Int J Biochem Cell Biol2004;36:1206-37.
69. Rincon-Choles H, Vasylyeva TL, Pergola PE, Bhandari B, Bhandari K, Zhang JH,Wang W, Gorin Y, Barnes JL, Abboud HE. ZO-1expression and phosphorylation indiabetic nephropathy. Diabetes2006;55:894-900.
70. Komarova YA, Mehta D, Malik AB. Dual regulation of endothelial junctionalpermeability. Sci STKE2007;2007:re8.
71. Vestweber D. VE-cadherin: the major endothelial adhesion molecule controllingcellular junctions and blood vessel formation. Arterioscler Thromb Vasc Biol2008;28:223-32.
72. Weis S, Shintani S, Weber A, Kirchmair R, Wood M, Cravens A, McSharry H,Iwakura A, Yoon YS, Himes N, Burstein D, Doukas J, Soll R, Losordo D, ChereshD. Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissueinjury following myocardial infarction. J Clin Invest2004;113:885-94.
73. Gong P, Angelini DJ, Yang S, Xia G, Cross AS, Mann D, Bannerman DD, Vogel SN,Goldblum SE. TLR4signaling is coupled to SRC family kinase activation, tyrosinephosphorylation of zonula adherens proteins, and opening of the paracellularpathway in human lung microvascular endothelia. J Biol Chem2008;283:13437-49.
74. van Buul JD, Anthony EC, Fernandez-Borja M, Burridge K, Hordijk PL.Proline-rich tyrosine kinase2(Pyk2) mediates vascular endothelial-cadherin-basedcell-cell adhesion by regulating beta-catenin tyrosine phosphorylation. J Biol Chem2005;280:21129-36.
75. Lambeng N, Wallez Y, Rampon C, Cand F, Christe G, Gulino-Debrac D, Vilgrain I,Huber P. Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic andquiescent adult tissues. Circ Res2005;96:384-91.
76. Biswas P, Zhang J, Schoenfeld JD, Schoenfeld D, Gratzinger D, Canosa S, MadriJA. Identification of the regions of PECAM-1involved in beta-and gamma-cateninassociations. Biochem Biophys Res Commun2005;329:1225-33.
77. Biswas P, Canosa S, Schoenfeld D, Schoenfeld J, Li P, Cheas LC, Zhang J,Cordova A, Sumpio B, Madri JA. PECAM-1affects GSK-3beta-mediatedbeta-catenin phosphorylation and degradation. Am J Pathol2006;169:314-24.
78. Garnacho C, Shuvaev V, Thomas A, McKenna L, Sun J, Koval M, Albelda S,Muzykantov V, Muro S. RhoA activation and actin reorganization involved inendothelial CAM-mediated endocytosis of anti-PECAM carriers: critical role fortyrosine686in the cytoplasmic tail of PECAM-1. Blood2008;111:3024-33.
79. Romer LH, Birukov KG, Garcia JG. Focal adhesions: paradigm for a signalingnexus. Circ Res2006;98:606-16.
80. Wu MH. Endothelial focal adhesions and barrier function. J Physiol2005;569:359-66.
81. Alexander JS, Zhu Y, Elrod JW, Alexander B, Coe L, Kalogeris TJ, Fuseler J.Reciprocal regulation of endothelial substrate adhesion and barrier function.Microcirculation2001;8:389-401.
82. Shikata Y, Birukov KG, Birukova AA, Verin A, Garcia JG. Involvement ofsite-specific FAK phosphorylation in sphingosine-1phosphate-andthrombin-induced focal adhesion remodeling: role of Src and GIT. Faseb J2003;17:2240-9.
83. Mehta D, Tiruppathi C, Sandoval R, Minshall RD, Holinstat M, Malik AB.Modulatory role of focal adhesion kinase in regulating human pulmonary arterialendothelial barrier function. J Physiol2002;539:779-89.
84. Quadri SK, Bhattacharjee M, Parthasarathi K, Tanita T, Bhattacharya J. Endothelialbarrier strengthening by activation of focal adhesion kinase. J Biol Chem2003;278:13342-9.
85. Quadri SK, Bhattacharya J. Resealing of endothelial junctions by focal adhesionkinase. Am J Physiol Lung Cell Mol Physiol2007;292:L334-42.
86. Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR,English D. Sphingosine1-phosphate promotes endothelial cell barrier integrity byEdg-dependent cytoskeletal rearrangement. J Clin Invest2001;108:689-701.
87. Singleton PA, Dudek SM, Chiang ET, Garcia JG. Regulation of sphingosine1-phosphate-induced endothelial cytoskeletal rearrangement and barrierenhancement by S1P1receptor, PI3kinase, Tiam1/Rac1, and alpha-actinin. Faseb J2005;19:1646-56.
88. Singleton PA, Dudek SM, Ma SF, Garcia JG. Transactivation of sphingosine1-phosphate receptors is essential for vascular barrier regulation. Novel role forhyaluronan and CD44receptor family. J Biol Chem2006;281:34381-93.
89. van Nieuw Amerongen GP, van Hinsbergh VW. Targets for pharmacologicalintervention of endothelial hyperpermeability and barrier function. VasculPharmacol2002;39:257-72.
90. Eum SY, Lee YW, Hennig B, Toborek M. VEGF regulates PCB104-mediatedstimulation of permeability and transmigration of breast cancer cells in humanmicrovascular endothelial cells. Exp Cell Res2004;296:231-44.
91. Shrivastava-Ranjan P, Rollin PE, Spiropoulou CF. Andes virus disrupts theendothelial cell barrier by induction of vascular endothelial growth factor anddownregulation of VE-cadherin. J Virol2010;84:11227-34.
92. Gorbunova E, Gavrilovskaya IN, Mackow ER. Pathogenic hantaviruses Andesvirus and Hantaan virus induce adherens junction disassembly by directing vascularendothelial cadherin internalization in human endothelial cells. J Virol2010;84:7405-11.
93.蒋伟，杨栋强，潘蕾，于海涛，李彧，王伟，王平忠，白雪帆.运用流式细胞术快速检测汉滩病毒滴度.细胞与分子免疫学杂志2011;27:579-581.
94.杨为松.肾综合征出血热.1999;第二版.
95. Carr JM, Hocking H, Bunting K, Wright PJ, Davidson A, Gamble J, Burrell CJ, LiP. Supernatants from dengue virus type-2infected macrophages inducepermeability changes in endothelial cell monolayers. J Med Virol2003;69:521-8.
96. Fuse C, Ishida Y, Hikita T, Asai T, Oku N. Junctional adhesion molecule-Cpromotes metastatic potential of HT1080human fibrosarcoma. J Biol Chem2007;282:8276-83.
97. Zachary I, Gliki G. Signaling transduction mechanisms mediating biologicalactions of the vascular endothelial growth factor family. Cardiovasc Res2001;49:568-81.
98. Wang W, Dentler WL, Borchardt RT. VEGF increases BMEC monolayerpermeability by affecting occludin expression and tight junction assembly. Am JPhysiol Heart Circ Physiol2001;280:H434-40.
99. Weis S, Cui J, Barnes L, Cheresh D. Endothelial barrier disruption byVEGF-mediated Src activity potentiates tumor cell extravasation and metastasis. JCell Biol2004;167:223-9.
100. Soldi R, Mitola S, Strasly M, Defilippi P, Tarone G, Bussolino F. Role ofalphavbeta3integrin in the activation of vascular endothelial growth factorreceptor-2. Embo J1999;18:882-92.
101. Geiger B, Bershadsky A, Pankov R, Yamada KM. Transmembrane crosstalkbetween the extracellular matrix--cytoskeleton crosstalk. Nat Rev Mol Cell Biol2001;2:793-805.
102. Woodard AS, Garcia-Cardena G, Leong M, Madri JA, Sessa WC, Languino LR.The synergistic activity of alphavbeta3integrin and PDGF receptor increases cellmigration. J Cell Sci1998;111(Pt4):469-78.
103. Orr FW, Wang HH, Lafrenie RM, Scherbarth S, Nance DM. Interactions betweencancer cells and the endothelium in metastasis. J Pathol2000;190:310-29.
104. Laferriere J, Houle F, Taher MM, Valerie K, Huot J. Transendothelial migration ofcolon carcinoma cells requires expression of E-selectin by endothelial cells andactivation of stress-activated protein kinase-2(SAPK2/p38) in the tumor cells. JBiol Chem2001;276:33762-72.
105. Holinstat M, Knezevic N, Broman M, Samarel AM, Malik AB, Mehta D.Suppression of RhoA activity by focal adhesion kinase-induced activation ofp190RhoGAP: role in regulation of endothelial permeability. J Biol Chem2006;281:2296-305.
106. Adamson RH, Curry FE, Adamson G, Liu B, Jiang Y, Aktories K, Barth H,Daigeler A, Golenhofen N, Ness W, Drenckhahn D. Rho and rho kinase modulationof barrier properties: cultured endothelial cells and intact microvessels of rats andmice. J Physiol2002;539:295-308.
107. Xu R, Feng X, Xie X, Zhang J, Wu D, Xu L. HIV-1Tat protein increases thepermeability of brain endothelial cells by both inhibiting occludin expression andcleaving occludin via matrix metalloproteinase-9. Brain Res2012;1436:13-9.
108. Partridge CA, Jeffrey JJ, Malik AB. A96-kDa gelatinase induced by TNF-alphacontributes to increased microvascular endothelial permeability. Am J Physiol1993;265:L438-47.
109. Loirand G, Guerin P, Pacaud P. Rho kinases in cardiovascular physiology andpathophysiology. Circ Res2006;98:322-34.
110.宋静，郭惠玲，王大江，余继锋，杜改萍，王晓奎，黄一飞.原子力显微镜观测小分子化合物J2对大鼠角膜移植术后内皮细胞膜表面的影响.中华眼科杂志2011;47:404-409.
111.朱杰.原子力显微镜在分子细胞生物学研究中的应用.现代物理知识2006;18:36-37.
2. Raftery MJ, Kraus AA, Ulrich R, Kruger DH, Schonrich G. Hantavirus infection ofdendritic cells. J Virol2002;76:10724-33.
3. Wang M, Wang J, Zhu Y, Xu Z, Yang K, Yang A, Jin B. Cellular immune responseto Hantaan virus nucleocapsid protein in the acute phase of hemorrhagic fever withrenal syndrome: correlation with disease severity. J Infect Dis2009;199:188-95.
4. Urbani S, Amadei B, Fisicaro P, Tola D, Orlandini A, Sacchelli L, Mori C, MissaleG, Ferrari C. Outcome of acute hepatitis C is related to virus-specific CD4functionand maturation of antiviral memory CD8responses. Hepatology2006;44:126-39.
5. Dvorak HF. Discovery of vascular permeability factor (VPF). Exp Cell Res2006;312:522-6.
6. Srikiatkhachorn A, Ajariyakhajorn C, Endy TP, Kalayanarooj S, Libraty DH, GreenS, Ennis FA, Rothman AL. Virus-induced decline in soluble vascular endothelialgrowth receptor2is associated with plasma leakage in dengue hemorrhagic Fever.J Virol2007;81:1592-600.
7. Wallez Y, Huber P. Endothelial adherens and tight junctions in vascularhomeostasis, inflammation and angiogenesis. Biochim Biophys Acta2008;1778:794-809.
8. Niikura M, Maeda A, Ikegami T, Saijo M, Kurane I, Morikawa S. Modification ofendothelial cell functions by Hantaan virus infection: prolonged hyper-permeabilityinduced by TNF-alpha of hantaan virus-infected endothelial cell monolayers. ArchVirol2004;149:1279-92.
9. Oelschlegel R, Kruger DH, Rang A. MxA-independent inhibition of Hantaan virusreplication induced by type I and type II interferon in vitro. Virus Res2007;127:100-5.
10.王平忠，李宜川，陈延平，等.肾综合征出血热患者TNF-α、IL-6、IL-4、 IFN-γ水平变化及意义.西南国防医药2007;17:396-398.
11. Wimer BM. Implications of the analogy between recombinant cytokine toxicitiesand manifestations of hantavirus infections. Cancer Biother Radiopharm1998;13:193-207.
12.王平忠，王九平，王雅格，等.肾综合征出血热患者IL-8、IP-10和RANTES的水平变化及意义.中国现代医学杂志2008;18:1248-1250.
13. Liu JM, Zhu Y, Xu ZW, Ouyang WM, Wang JP, Liu XS, Cao YX, Li Q, Fang L,Zhuang R, Yang AG, Jin BQ. Dynamic changes of apoptosis-inducing ligands andTh1/Th2like subpopulations in Hantaan virus-induced hemorrhagic fever withrenal syndrome. Clin Immunol2006;119:245-51.
14. Mou DL, Wang YP, Huang CX, Li GY, Pan L, Yang WS, Bai XF. Cellular entry ofHantaan virus A9strain: specific interactions with beta3integrins and a novel70kDa protein. Biochem Biophys Res Commun2006;339:611-7.
15.牟丹蕾，白雪帆*，黄长形，等.人整合素αvβ3在CHO细胞表达的研究.中华微生物学和免疫学杂志200323:618-624.
16. Switala-Jelen K, Dabrowska K, Opolski A, Lipinska L, Nowaczyk M, Gorski A.The biological functions of beta3integrins. Folia Biol (Praha)2004;50:143-52.
17. Luo BH, Springer TA. Integrin structures and conformational signaling. Curr OpinCell Biol2006;18:579-86.
18. Mould AP, Travis MA, Barton SJ, Hamilton JA, Askari JA, Craig SE, MacdonaldPR, Kammerer RA, Buckley PA, Humphries MJ. Evidence that monoclonalantibodies directed against the integrin beta subunit plexin/semaphorin/integrindomain stimulate function by inducing receptor extension. J Biol Chem2005;280:4238-46.
19. Raymond T, Gorbunova E, Gavrilovskaya IN, Mackow ER. Pathogenichantaviruses bind plexin-semaphorin-integrin domains present at the apex ofinactive, bent alphavbeta3integrin conformers. Proc Natl Acad Sci U S A2005;102:1163-8.
20. Zhang JL, Wang JL, Gao N, Chen ZT, Tian YP, An J. Up-regulated expression ofbeta3integrin induced by dengue virus serotype2infection associated with virusentry into human dermal microvascular endothelial cells. Biochem Biophys ResCommun2007;356:763-8.
21. Tang QH, Zhang YM, Xu YZ, He L, Dai C, Sun P. Up-regulation of integrin beta3expression in porcine vascular endothelial cells cultured in vitro by classical swinefever virus. Vet Immunol Immunopathol2010;133:237-42.
22. Gahmberg CG FS, Nurmi SM. Regulation of integrin activity and signaling.Biochim Biophysica Acta2009;1790:431-444.
23. Mahabeleshwar GH, Feng W, Phillips DR, Byzova TV. Integrin signaling is criticalfor pathological angiogenesis. J Exp Med2006;203:2495-507.
24. Gahmberg CG, Fagerholm SC, Nurmi SM, Chavakis T, Marchesan S, Gronholm M.Regulation of integrin activity and signalling. Biochim Biophys Acta2009;1790:431-44.
25. Martin KH, Slack JK, Boerner SA, Martin CC, Parsons JT. Integrin connectionsmap: to infinity and beyond. Science2002;296:1652-3.
26. Kumar CC. Signaling by integrin receptors. Oncogene1998;17:1365-73.
27. Yanagihara R, Silverman DJ. Experimental infection of human vascular endothelialcells by pathogenic and nonpathogenic hantaviruses. Arch Virol1990;111:281-6.
28. Gavrilovskaya IN, Shepley M, Shaw R, Ginsberg MH, Mackow ER. beta3Integrins mediate the cellular entry of hantaviruses that cause respiratory failure.Proc Natl Acad Sci U S A1998;95:7074-9.
29. Zaki SR, Greer PW, Coffield LM, Goldsmith CS, Nolte KB, Foucar K, FeddersenRM, Zumwalt RE, Miller GL, Khan AS, et al. Hantavirus pulmonary syndrome.Pathogenesis of an emerging infectious disease. Am J Pathol1995;146:552-79.
30. Gavrilovskaya IN, Peresleni T, Geimonen E, Mackow ER. Pathogenic hantavirusesselectively inhibit beta3integrin directed endothelial cell migration. Arch Virol2002;147:1913-31.
31. Gavrilovskaya IN, Gorbunova EE, Mackow NA, Mackow ER. Hantaviruses directendothelial cell permeability by sensitizing cells to the vascular permeability factorVEGF, while angiopoietin1and sphingosine1-phosphate inhibithantavirus-directed permeability. J Virol2008;82:5797-806.
32. Gavard J, Patel V, Gutkind JS. Angiopoietin-1prevents VEGF-induced endothelialpermeability by sequestering Src through mDia. Dev Cell2008;14:25-36.
33. Lampugnani MG, Dejana E. Adherens junctions in endothelial cells regulate vesselmaintenance and angiogenesis. Thromb Res2007;120Suppl2:S1-6.
34. Mackow ER, Gavrilovskaya IN. Hantavirus regulation of endothelial cell functions.Thromb Haemost2009;102:1030-41.
35. Buranda T, Wu Y, Perez D, Jett SD, BonduHawkins V, Ye C, Edwards B, Hall P,Larson RS, Lopez GP, Sklar LA, Hjelle B. Recognition of decay accelerating factorand alpha(v)beta(3) by inactivated hantaviruses: Toward the development ofhigh-throughput screening flow cytometry assays. Anal Biochem2010;402:151-60.
36. Krautkramer E, Zeier M. Hantavirus causing hemorrhagic fever with renalsyndrome enters from the apical surface and requires decay-accelerating factor(DAF/CD55). J Virol2008;82:4257-64.
37. Choi Y, Kwon YC, Kim SI, Park JM, Lee KH, Ahn BY. A hantavirus causinghemorrhagic fever with renal syndrome requires gC1qR/p32for efficient cellbinding and infection. Virology2008;381:178-83.
38.徐芳玲，杨占秋.汉坦病毒受体.病毒学报2004;20:288-290.
39. Ogino M, Yoshimatsu K, Ebihara H, Arikawa J. N-acetylgalactosamine(GalNAc)-specific lectins mediate enhancement of Hantaan virus infection. ArchVirol1999;144:1765-77.
40. Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability.Physiol Rev2006;86:279-367.
41. Vestweber D, Winderlich M, Cagna G, Nottebaum AF. Cell adhesion dynamics atendothelial junctions: VE-cadherin as a major player. Trends Cell Biol2009;19:8-15.
42. Harris ES NW. VE-cadherin: at the front, center, and sides of endothelial cellorganization and function. Curr Opin Cell Biol2010;22651-8.
43.赵克森，黄巧冰.血管通透性增高的基本机制.中国病理生理杂志2003;19:549-553.
44.李彧，王伟，王平忠.肾综合征出血热外周静脉血细胞因子的变化与病情的关系.中国病毒病杂志2011;1:260-263.
45. Peterson LJ, Rajfur Z, Maddox AS, Freel CD, Chen Y, Edlund M, Otey C, BurridgeK. Simultaneous stretching and contraction of stress fibers in vivo. Mol Biol Cell2004;15:3497-508.
46. Thiagarajan P, Le A, Snuggs MB, VanWinkle B. The role of carboxy-terminalglycosaminoglycan-binding domain of vitronectin in cytoskeletal organization andmigration of endothelial cells. Cell Adhes Commun1996;4:317-25.
47. Lee TY, Gotlieb AI. Microfilaments and microtubules maintain endothelial integrity.Microsc Res Tech2003;60:115-27.
48. Jacobson JR, Garcia JG. Novel therapies for microvascular permeability in sepsis.Curr Drug Targets2007;8:509-14.
49. Bogatcheva NV, Verin AD. The role of cytoskeleton in the regulation of vascularendothelial barrier function. Microvasc Res2008;76:202-7.
50. Bogatcheva NV, Verin AD, Wang P, Birukova AA, Birukov KG, Mirzopoyazova T,Adyshev DM, Chiang ET, Crow MT, Garcia JG. Phorbol esters increase MLCphosphorylation and actin remodeling in bovine lung endothelium withoutincreased contraction. Am J Physiol Lung Cell Mol Physiol2003;285:L415-26.
51. Tiruppathi C, Minshall RD, Paria BC, Vogel SM, Malik AB. Role of Ca2+signaling in the regulation of endothelial permeability. Vascul Pharmacol2002;39:173-85.
52. van Nieuw Amerongen GP, van Hinsbergh VW. Endogenous RhoA inhibitorprotects endothelial barrier. Circ Res2007;101:7-9.
53. Gorovoy M, Neamu R, Niu J, Vogel S, Predescu D, Miyoshi J, Takai Y, Kini V,Mehta D, Malik AB, Voyno-Yasenetskaya T. RhoGDI-1modulation of the activityof monomeric RhoGTPase RhoA regulates endothelial barrier function in mouselungs. Circ Res2007;101:50-8.
54. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature2002;420:629-35.
55. Wojciak-Stothard B, Ridley AJ. Rho GTPases and the regulation of endothelialpermeability. Vascul Pharmacol2002;39:187-99.
56. Ivanov AI, Parkos CA, Nusrat A. Cytoskeletal regulation of epithelial barrierfunction during inflammation. Am J Pathol2010;177:512-24.
57. Amano M, Nakayama M, Kaibuchi K. Rho-kinase/ROCK: A key regulator of thecytoskeleton and cell polarity. Cytoskeleton (Hoboken)2010;67:545-54.
58. Hirano S, Rees RS, Yancy SL, Welsh MJ, Remick DG, Yamada T, Hata J, GilmontRR. Endothelial barrier dysfunction caused by LPS correlates with phosphorylationof HSP27in vivo. Cell Biol Toxicol2004;20:1-14.
59. Tar K, Csortos C, Czikora I, Olah G, Ma SF, Wadgaonkar R, Gergely P, Garcia JG,Verin AD. Role of protein phosphatase2A in the regulation of endothelial cellcytoskeleton structure. J Cell Biochem2006;98:931-53.
60. Borbiev T, Birukova A, Liu F, Nurmukhambetova S, Gerthoffer WT, Garcia JG,Verin AD. p38MAP kinase-dependent regulation of endothelial cell permeability.Am J Physiol Lung Cell Mol Physiol2004;287:L911-8.
61. Bogatcheva NV, Birukova A, Borbiev T, Kolosova I, Liu F, Garcia JG, Verin AD.Caldesmon is a cytoskeletal target for PKC in endothelium. J Cell Biochem2006;99:1593-605.
62. Lee YH, Kayyali US, Sousa AM, Rajan T, Lechleider RJ, Day RM. Transforminggrowth factor-beta1effects on endothelial monolayer permeability involve focaladhesion kinase/Src. Am J Respir Cell Mol Biol2007;37:485-93.
63. Bogatcheva NV, Adyshev D, Mambetsariev B, Moldobaeva N, Verin AD.Involvement of microtubules, p38, and Rho kinases pathway in2-methoxyestradiol-induced lung vascular barrier dysfunction. Am J Physiol LungCell Mol Physiol2007;292:L487-99.
64. Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, Garcia JG.Microtubule disassembly increases endothelial cell barrier dysfunction: role ofMLC phosphorylation. Am J Physiol Lung Cell Mol Physiol2001;281:L565-74.
65. Birukova AA, Adyshev D, Gorshkov B, Bokoch GM, Birukov KG, Verin AD.GEF-H1is involved in agonist-induced human pulmonary endothelial barrierdysfunction. Am J Physiol Lung Cell Mol Physiol2006;290:L540-8.
66. Sehrawat S, Cullere X, Patel S, Italiano J, Jr., Mayadas TN. Role of Epac1, anexchange factor for Rap GTPases, in endothelial microtubule dynamics and barrierfunction. Mol Biol Cell2008;19:1261-70.
67. Gonzalez-Mariscal L, Tapia R, Chamorro D. Crosstalk of tight junctioncomponents with signaling pathways. Biochim Biophys Acta2008;1778:729-56.
68. Harhaj NS, Antonetti DA. Regulation of tight junctions and loss of barrier functionin pathophysiology. Int J Biochem Cell Biol2004;36:1206-37.
69. Rincon-Choles H, Vasylyeva TL, Pergola PE, Bhandari B, Bhandari K, Zhang JH,Wang W, Gorin Y, Barnes JL, Abboud HE. ZO-1expression and phosphorylation indiabetic nephropathy. Diabetes2006;55:894-900.
70. Komarova YA, Mehta D, Malik AB. Dual regulation of endothelial junctionalpermeability. Sci STKE2007;2007:re8.
71. Vestweber D. VE-cadherin: the major endothelial adhesion molecule controllingcellular junctions and blood vessel formation. Arterioscler Thromb Vasc Biol2008;28:223-32.
72. Weis S, Shintani S, Weber A, Kirchmair R, Wood M, Cravens A, McSharry H,Iwakura A, Yoon YS, Himes N, Burstein D, Doukas J, Soll R, Losordo D, ChereshD. Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissueinjury following myocardial infarction. J Clin Invest2004;113:885-94.
73. Gong P, Angelini DJ, Yang S, Xia G, Cross AS, Mann D, Bannerman DD, Vogel SN,Goldblum SE. TLR4signaling is coupled to SRC family kinase activation, tyrosinephosphorylation of zonula adherens proteins, and opening of the paracellularpathway in human lung microvascular endothelia. J Biol Chem2008;283:13437-49.
74. van Buul JD, Anthony EC, Fernandez-Borja M, Burridge K, Hordijk PL.Proline-rich tyrosine kinase2(Pyk2) mediates vascular endothelial-cadherin-basedcell-cell adhesion by regulating beta-catenin tyrosine phosphorylation. J Biol Chem2005;280:21129-36.
75. Lambeng N, Wallez Y, Rampon C, Cand F, Christe G, Gulino-Debrac D, Vilgrain I,Huber P. Vascular endothelial-cadherin tyrosine phosphorylation in angiogenic andquiescent adult tissues. Circ Res2005;96:384-91.
76. Biswas P, Zhang J, Schoenfeld JD, Schoenfeld D, Gratzinger D, Canosa S, MadriJA. Identification of the regions of PECAM-1involved in beta-and gamma-cateninassociations. Biochem Biophys Res Commun2005;329:1225-33.
77. Biswas P, Canosa S, Schoenfeld D, Schoenfeld J, Li P, Cheas LC, Zhang J,Cordova A, Sumpio B, Madri JA. PECAM-1affects GSK-3beta-mediatedbeta-catenin phosphorylation and degradation. Am J Pathol2006;169:314-24.
78. Garnacho C, Shuvaev V, Thomas A, McKenna L, Sun J, Koval M, Albelda S,Muzykantov V, Muro S. RhoA activation and actin reorganization involved inendothelial CAM-mediated endocytosis of anti-PECAM carriers: critical role fortyrosine686in the cytoplasmic tail of PECAM-1. Blood2008;111:3024-33.
79. Romer LH, Birukov KG, Garcia JG. Focal adhesions: paradigm for a signalingnexus. Circ Res2006;98:606-16.
80. Wu MH. Endothelial focal adhesions and barrier function. J Physiol2005;569:359-66.
81. Alexander JS, Zhu Y, Elrod JW, Alexander B, Coe L, Kalogeris TJ, Fuseler J.Reciprocal regulation of endothelial substrate adhesion and barrier function.Microcirculation2001;8:389-401.
82. Shikata Y, Birukov KG, Birukova AA, Verin A, Garcia JG. Involvement ofsite-specific FAK phosphorylation in sphingosine-1phosphate-andthrombin-induced focal adhesion remodeling: role of Src and GIT. Faseb J2003;17:2240-9.
83. Mehta D, Tiruppathi C, Sandoval R, Minshall RD, Holinstat M, Malik AB.Modulatory role of focal adhesion kinase in regulating human pulmonary arterialendothelial barrier function. J Physiol2002;539:779-89.
84. Quadri SK, Bhattacharjee M, Parthasarathi K, Tanita T, Bhattacharya J. Endothelialbarrier strengthening by activation of focal adhesion kinase. J Biol Chem2003;278:13342-9.
85. Quadri SK, Bhattacharya J. Resealing of endothelial junctions by focal adhesionkinase. Am J Physiol Lung Cell Mol Physiol2007;292:L334-42.
86. Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR,English D. Sphingosine1-phosphate promotes endothelial cell barrier integrity byEdg-dependent cytoskeletal rearrangement. J Clin Invest2001;108:689-701.
87. Singleton PA, Dudek SM, Chiang ET, Garcia JG. Regulation of sphingosine1-phosphate-induced endothelial cytoskeletal rearrangement and barrierenhancement by S1P1receptor, PI3kinase, Tiam1/Rac1, and alpha-actinin. Faseb J2005;19:1646-56.
88. Singleton PA, Dudek SM, Ma SF, Garcia JG. Transactivation of sphingosine1-phosphate receptors is essential for vascular barrier regulation. Novel role forhyaluronan and CD44receptor family. J Biol Chem2006;281:34381-93.
89. van Nieuw Amerongen GP, van Hinsbergh VW. Targets for pharmacologicalintervention of endothelial hyperpermeability and barrier function. VasculPharmacol2002;39:257-72.
90. Eum SY, Lee YW, Hennig B, Toborek M. VEGF regulates PCB104-mediatedstimulation of permeability and transmigration of breast cancer cells in humanmicrovascular endothelial cells. Exp Cell Res2004;296:231-44.
91. Shrivastava-Ranjan P, Rollin PE, Spiropoulou CF. Andes virus disrupts theendothelial cell barrier by induction of vascular endothelial growth factor anddownregulation of VE-cadherin. J Virol2010;84:11227-34.
92. Gorbunova E, Gavrilovskaya IN, Mackow ER. Pathogenic hantaviruses Andesvirus and Hantaan virus induce adherens junction disassembly by directing vascularendothelial cadherin internalization in human endothelial cells. J Virol2010;84:7405-11.
93.蒋伟，杨栋强，潘蕾，于海涛，李彧，王伟，王平忠，白雪帆.运用流式细胞术快速检测汉滩病毒滴度.细胞与分子免疫学杂志2011;27:579-581.
94.杨为松.肾综合征出血热.1999;第二版.
95. Carr JM, Hocking H, Bunting K, Wright PJ, Davidson A, Gamble J, Burrell CJ, LiP. Supernatants from dengue virus type-2infected macrophages inducepermeability changes in endothelial cell monolayers. J Med Virol2003;69:521-8.
96. Fuse C, Ishida Y, Hikita T, Asai T, Oku N. Junctional adhesion molecule-Cpromotes metastatic potential of HT1080human fibrosarcoma. J Biol Chem2007;282:8276-83.
97. Zachary I, Gliki G. Signaling transduction mechanisms mediating biologicalactions of the vascular endothelial growth factor family. Cardiovasc Res2001;49:568-81.
98. Wang W, Dentler WL, Borchardt RT. VEGF increases BMEC monolayerpermeability by affecting occludin expression and tight junction assembly. Am JPhysiol Heart Circ Physiol2001;280:H434-40.
99. Weis S, Cui J, Barnes L, Cheresh D. Endothelial barrier disruption byVEGF-mediated Src activity potentiates tumor cell extravasation and metastasis. JCell Biol2004;167:223-9.
100. Soldi R, Mitola S, Strasly M, Defilippi P, Tarone G, Bussolino F. Role ofalphavbeta3integrin in the activation of vascular endothelial growth factorreceptor-2. Embo J1999;18:882-92.
101. Geiger B, Bershadsky A, Pankov R, Yamada KM. Transmembrane crosstalkbetween the extracellular matrix--cytoskeleton crosstalk. Nat Rev Mol Cell Biol2001;2:793-805.
102. Woodard AS, Garcia-Cardena G, Leong M, Madri JA, Sessa WC, Languino LR.The synergistic activity of alphavbeta3integrin and PDGF receptor increases cellmigration. J Cell Sci1998;111(Pt4):469-78.
103. Orr FW, Wang HH, Lafrenie RM, Scherbarth S, Nance DM. Interactions betweencancer cells and the endothelium in metastasis. J Pathol2000;190:310-29.
104. Laferriere J, Houle F, Taher MM, Valerie K, Huot J. Transendothelial migration ofcolon carcinoma cells requires expression of E-selectin by endothelial cells andactivation of stress-activated protein kinase-2(SAPK2/p38) in the tumor cells. JBiol Chem2001;276:33762-72.
105. Holinstat M, Knezevic N, Broman M, Samarel AM, Malik AB, Mehta D.Suppression of RhoA activity by focal adhesion kinase-induced activation ofp190RhoGAP: role in regulation of endothelial permeability. J Biol Chem2006;281:2296-305.
106. Adamson RH, Curry FE, Adamson G, Liu B, Jiang Y, Aktories K, Barth H,Daigeler A, Golenhofen N, Ness W, Drenckhahn D. Rho and rho kinase modulationof barrier properties: cultured endothelial cells and intact microvessels of rats andmice. J Physiol2002;539:295-308.
107. Xu R, Feng X, Xie X, Zhang J, Wu D, Xu L. HIV-1Tat protein increases thepermeability of brain endothelial cells by both inhibiting occludin expression andcleaving occludin via matrix metalloproteinase-9. Brain Res2012;1436:13-9.
108. Partridge CA, Jeffrey JJ, Malik AB. A96-kDa gelatinase induced by TNF-alphacontributes to increased microvascular endothelial permeability. Am J Physiol1993;265:L438-47.
109. Loirand G, Guerin P, Pacaud P. Rho kinases in cardiovascular physiology andpathophysiology. Circ Res2006;98:322-34.
110.宋静，郭惠玲，王大江，余继锋，杜改萍，王晓奎，黄一飞.原子力显微镜观测小分子化合物J2对大鼠角膜移植术后内皮细胞膜表面的影响.中华眼科杂志2011;47:404-409.
111.朱杰.原子力显微镜在分子细胞生物学研究中的应用.现代物理知识2006;18:36-37.
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