Rickettsiae induce microvascular hyperpermeability via phosphorylation of VE-cadherins: evidence from atomic force microscopy and biochemical studies.
Rickettsiae induce microvascular hyperpermeability via phosphorylation of VE-cadherins: evidence from atomic force microscopy and biochemical studies.
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人力病通过VE-钙粘着蛋白的磷酸化诱导微血管高温性：来自原子力显微镜和生化研究的证据。
DOI：
10.1371/journal.pntd.0001699
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发表时间：
2012
影响因子：
3.8
通讯作者：
Oberhauser A
中科院分区：
文献类型：
--
作者：
Gong B;Ma L;Liu Y;Gong Q;Shelite T;Bouyer D;Boor PJ;Lee YS;Oberhauser A
斑点热群(SFG)立克次体感染微血管内皮细胞(ECs)的最显著的病理生理效应是血管通透性增加，促进血管源性脑水肿和非心源性肺水肿，这是重症病例的大部分发病率和死亡率的原因。到目前为止，SFG立克次体增加EC通透性的细胞和分子机制很大程度上是未知的。在本研究中，我们使用原子力显微镜(AFM)来研究血管内皮细胞(VE)-钙粘蛋白与感染了蒙丹氏杆菌的人脑微血管内皮细胞之间的相互作用力。蒙丹氏杆菌在遗传上类似于立克次氏杆菌和康诺里杆菌，表现出类似的入侵细胞的能力，但是非致病的，可以在生物安全级别2(BSL2)的条件下进行实验操作。我们发现，感染的内皮细胞VE-钙粘蛋白-EC的相互作用显著减少。此外，我们应用免疫荧光染色、免疫沉淀磷酸化试验和体外内皮通透性试验研究了可能参与SFG立克次体感染的潜在病理改变的血管通透性增加的生化机制。一个重要的发现是，感染蒙古大黄后，VE-钙粘蛋白的酪氨酸磷酸化从48小时开始显著激活，72小时达到高峰。体外通透性测定显示72hP.I.微血管通透性增强。另一方面，AFM实验显示，在48小时P.I.时，VE-钙粘蛋白-EC相互作用力显著减少。我们的结论是，在感染SFG立克次体后，VE-钙粘附素的磷酸化直接减弱内皮细胞黏附连接处的亲水性蛋白质-蛋白质相互作用，并可能导致内皮细胞旁细胞屏障功能障碍，导致微血管高通透性。这些新的方法应该被证明在BSL3环境中表征抗原性相关的SFG康氏立克次体和立克次体是有用的。未来的研究可能会导致新的治疗策略的发展，以抑制SFG立克次体中VE-钙粘素相关的微血管高通透性。立克次体疾病是严重的人类传染病。一些斑点热群(SFG)立克次体病原体是生物恐怖因子。SFG立克次体病的一个主要临床特征是内皮细胞感染导致血管通透性增强。先前的研究表明，SFG立克次体引起剂量依赖性的高通透性，这与细胞间黏附连接(AJs)的破坏有关。在立克次体感染期间，连接复合体被破坏，最终导致内皮细胞旁环境发生变化的潜在分子机制仍然很大程度上尚不清楚。现有证据表明，炎症刺激可以触发AJs各种成分的酪氨酸磷酸化，主要是血管内皮细胞钙粘蛋白(VE-cadherin)。这会导致AJ的缝隙，部分原因是磷酸化导致质膜VE-钙粘附素的不稳定和内吞作用的增加，大大增加了细胞旁的渗漏。在这里，我们假设SFG立克次体感染诱导内皮细胞产生与酪氨酸残基磷酸化相关的VE-钙粘附素改变。利用原子力显微镜的纳米力学研究和主要AJ蛋白VE-钙粘蛋白的生化分析，我们推测VE-钙粘蛋白的磷酸化直接减弱了VE-钙粘蛋白之间的亲和性相互作用。该实验方法为立克次体感染的研究提供了一条新途径。这一策略在发现节肢动物传播的致命立克次体的新治疗策略方面应该被证明是有用的。
The most prominent pathophysiological effect of spotted fever group (SFG) rickettsial infection of microvascular endothelial cells (ECs) is an enhanced vascular permeability, promoting vasogenic cerebral edema and non-cardiogenic pulmonary edema, which are responsible for most of the morbidity and mortality in severe cases. To date, the cellular and molecular mechanisms by which SFG Rickettsia increase EC permeability are largely unknown. In the present study we used atomic force microscopy (AFM) to study the interactive forces between vascular endothelial (VE)-cadherin and human cerebral microvascular EC infected with R. montanensis, which is genetically similar to R. rickettsii and R. conorii, and displays a similar ability to invade cells, but is non-pathogenic and can be experimentally manipulated under Biosafety Level 2 (BSL2) conditions. We found that infected ECs show a significant decrease in VE-cadherin-EC interactions. In addition, we applied immunofluorescent staining, immunoprecipitation phosphorylation assay, and an in vitro endothelial permeability assay to study the biochemical mechanisms that may participate in the enhanced vascular permeability as an underlying pathologic alteration of SFG rickettsial infection. A major finding is that infection of R. montanensis significantly activated tyrosine phosphorylation of VE-cadherin beginning at 48 hr and reaching a peak at 72 hr p.i. In vitro permeability assay showed an enhanced microvascular permeability at 72 hr p.i. On the other hand, AFM experiments showed a dramatic reduction in VE-cadherin-EC interactive forces at 48 hr p.i. We conclude that upon infection by SFG rickettsiae, phosphorylation of VE-cadherin directly attenuates homophilic protein–protein interactions at the endothelial adherens junctions, and may lead to endothelial paracellular barrier dysfunction causing microvascular hyperpermeability. These new approaches should prove useful in characterizing the antigenically related SFG rickettsiae R. conorii and R. rickettsii in a BSL3 environment. Future studies may lead to the development of new therapeutic strategies to inhibit the VE-cadherin-associated microvascular hyperpermeability in SFG rickettsioses. Rickettsial diseases are serious human infections. Some spotted fever group (SFG) rickettsial pathogens are bioterror agents. A major clinical hallmark of SFG rickettsial disease is the infection of endothelial cells leading to enhanced vascular permeability. Previous studies show that SFG rickettsiae cause dose-dependent hyperpermeability, which was associated with disruption of intercellular adherens junctions (AJs). The underlying molecular mechanism by which the junctional complexes are disrupted, ultimately causing changes in the endothelial paracellular milieu during rickettsial infection, remains largely unclear. The available evidence suggests that inflammatory stimuli can trigger tyrosine phosphorylation of various components of AJs, mainly the vascular endothelial–cadherin (VE-cadherin). This causes gaps at AJs, partially due to phosphorylation-induced destabilization of VE-cadherins at the plasma membrane and increased endocytosis, greatly increasing paracellular leaks. Here, we hypothesize that infection by SFG rickettsiae induces endothelial cells to develop altered VE-cadherin in association with phosphorylation of tyrosine residues. Utilizing nano-mechanical studies with atomic force microscopy and biochemical analysis of the major AJ protein VE-cadherin, we have implicated that phosphorylation of VE-cadherin directly attenuates homophilic interactions between VE-cadherins. The experimental approach advances a new way of studying rickettsial infection. This strategy should prove useful in uncovering novel therapeutic strategies for virulent arthropod-borne rickettsioses.
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影响因子：
4
作者：
Dejana, Elisabetta;Orsenigo, Fabrizio;Lampugnani, Maria Grazia
通讯作者：
Lampugnani, Maria Grazia
DOI：
10.1073/pnas.070052697
发表时间：
2000-04-11
影响因子：
11.1
作者：
Baumgartner, W;Hinterdorfer, P;Drenckhahn, D
通讯作者：
Drenckhahn, D
影响因子：
5.4
作者：
Gorbunova, Elena E.;Gavrilovskaya, Irina N.;Mackow, Erich R.
通讯作者：
Mackow, Erich R.
影响因子：
17.1
作者：
Alsteens, David;Dupres, Vincent;Klotz, Stephen A.;Gaur, Nand K.;Lipke, Peter N.;Dufrene, Yves F.
通讯作者：
Dufrene, Yves F.
DOI：
10.1073/pnas.96.17.9815
发表时间：
1999-08-17
影响因子：
11.1
作者：
Corada, M;Mariotti, M;Dejana, E
通讯作者：
Dejana, E