Compartmentalized, functional role of angiogenin during spotted fever group rickettsia-induced endothelial barrier dysfunction: evidence of possible mediation by host tRNA-derived small noncoding RNAs.
Compartmentalized, functional role of angiogenin during spotted fever group rickettsia-induced endothelial barrier dysfunction: evidence of possible mediation by host tRNA-derived small noncoding RNAs.
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DOI：
10.1186/1471-2334-13-285
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发表时间：
2013-06-23
影响因子：
3.7
通讯作者：
Walker DH
中科院分区：
文献类型：
--
作者：
Gong B;Lee YS;Lee I;Shelite TR;Kunkeaw N;Xu G;Lee K;Jeon SH;Johnson BH;Chang Q;Ha T;Mendell NL;Cheng X;Bouyer DH;Boor PJ;Ksiazek TG;Walker DH
微血管内皮屏障功能障碍是斑疹热组（SFG）立克次体病的中心谜团。血管生成素（Angiogenin， ANG）是最早发现的血管生成因子之一，其中一些与作为内皮粘附蛋白的VE-cadherins的磷酸化有关。尽管已知外源性ANG会转运到生长中的内皮细胞（ECs）的细胞核中，并在其中发挥功能作用，但在静止的内皮细胞中未检测到核ANG。除了核作用外，ANG还被认为在细胞质中发挥作用，因为它的RNase活性可以裂解tRNA产生小rna。最近，这种trna衍生的RNA片段（trf）已被证明是在应激条件下诱导的。所有这些观察结果提出了一个有趣的假设，即ANG在立克次体感染后被诱导并产生可能在SFG立克次体病中起作用的tRFs。C3H/HeN小鼠静脉注射亚致死剂量的conconi。在感染后第1,3,5天（p.i），收集肝脏、肺和脑进行conorii和血管生成素（ANG）的免疫荧光（IF）研究。将人脐静脉内皮细胞（HUVECs）分别感染conorii弧菌24、48、72小时后，用1μg/ml重组人ANG （rANG）在正常培养基中孵育2小时。HUVEC样品进行IF、外源性ANG易位、内皮通透性和免疫沉淀磷酸化测定。为了鉴定立克次体感染后的小分子非编码rna (sncRNAs)，使用Illumina 2000仪器对小鼠肺组织和HUVECs的rna进行文库制备和深度测序分析。鉴定出的sncrna通过Northern杂交得到确认，并使用BLAST和RNA杂交程序在计算机上预测其靶mrna。在目前的研究中，我们已经证实内皮细胞中ANG的上调与SFG立克次体感染在体内共定位。我们还提供了直接证据，表明立克次体感染使人ECs在感染后不同时间以区隔模式对外源性ANG易位敏感。通常，外源性ANG在感染后24小时转运到细胞核，72小时转运到细胞质。ANG细胞质易位增强了ve -钙粘蛋白的磷酸化和不稳定，减弱了内皮屏障功能。值得注意的是，深度测序分析检测到的trf主要来自宿主trna的5'-一半，是由ANG诱导的。Northern杂交在小鼠组织和人类细胞中验证了tRNA-ValGTG和tRNA-GlyGCC两个最丰富克隆的trf。生物信息学分析预测，这些tRFs可能与内皮屏障、宿主细胞炎症反应和自噬相关的转录物相互作用。我们的数据为分区ANG在SFG立克次体病中的作用提供了新的见解，并强调了其可能通过trf介导。
Microvascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses. C3H/HeN mice were infected intravenously with a sublethal dose of R. conorii. At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1μg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs. In the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in vivo. We also have provided direct evidence that rickettsial infection sensitizes human ECs to the translocation of exogenous ANG in a compartmentalized pattern at different times post-infection. Typically, exogenous ANG translocates into the nucleus at 24 hrs and to the cytoplasm at 72 hrs post-infection. The ANG cytoplasmic translocation enhances phosphorylation and destabilization of VE-cadherin and attenuates endothelial barrier function. Of note, deep sequencing analysis detected tRFs, mostly derived from the 5'-halves of host tRNAs, that are induced by ANG. Northern hybridization validates the two most abundantly cloned tRFs derived from tRNA-ValGTG and tRNA-GlyGCC, in both mouse tissues and human cells. Bioinformatics analysis predicted that these tRFs may interact with transcripts associated with the endothelial barrier, the host cell inflammatory response, and autophagy. Our data provide new insight into the role of compartmentalized ANG during SFG rickettsioses, and highlight its possible mediation through tRFs.
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作者：
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DOI：
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