DYMANICS OF ENDOTHELIAL CELL SIGNALING AND SIVE NEUROINFLAMMATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 猴免疫缺陷病毒(SIV)对血脑屏障通透性的影响可能是由中枢神经系统对病毒存在的胶质反应启动的。这种针对病毒的局部炎症反应通过RNA调节和蛋白质磷酸化来驱动内皮细胞的失调。我们通过识别和量化SIV诱导的促炎通路和由此导致的内皮细胞内信息水平的变化来检查内皮细胞间信号转导，以响应SIV。我们使用了共聚焦显微镜、细胞因子多重分析、RNA微阵列和RT-PCR。我们确定了一种新的下调CD263的方法，CD263是SIV刺激的内皮细胞中的一种肿瘤坏死因子诱骗受体，当内皮细胞遇到促炎刺激时，CD263通常用于减轻TRAIL的损害效应。受SIVmac251感染的巨噬细胞的上清液足以使星形胶质细胞过度产生约4倍的肿瘤坏死因子。此外，同样的培养上清液增加了内皮细胞中CCL2和CXCL8的产生。我们利用RNA微阵列技术对SIVmac251感染引起的猕猴血管内皮细胞的RNA调控进行了研究。这些研究证实，内皮细胞的炎症/激活是由促炎细胞因子，尤其是肿瘤坏死因子驱动的。内皮细胞中的肿瘤坏死因子受体被其配体激活，导致内皮细胞中核因子B的激活。这导致随后MLCK的上调/磷酸化，肌动蛋白细胞骨架的重组，以及ZO-1的重新分布。ZO-1的移位导致内皮紧密连接功能障碍，可能是通过JAM-2和claudin 8介导的。我们能够通过针对MLCK和FAK的抑制剂在很大程度上缓解SIVmac251驱动的原代脑微血管内皮细胞mRNA变化。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The effects of simian immunodeficiency virus (SIV) on blood-brain barrier permeability are likely initiated by glial response to the presence of virus in the central nervous system. This localized inflammatory reaction to the virus drives dysregulation of the endothelium by RNA modulation and protein phosphorylation. We examined interendothelial signaling by identifying and quantifying SIV-induced pro-inflammatory pathways and resultant message level changes in the endothelium, in response to SIV. We utilized confocal microscopy, cytokine multiplexing, RNA microarray, and RT-PCR. We determined a novel downregulation of CD263, a TNF decoy receptor in SIV stimulated endothelial cells, that normally serves to mitigate the damaging effects of TRAIL when the endothelium encounters an upsurge of proinflammatory stimuli. Supernatant from SIVmac251 infected macrophage was sufficient to cause astrocytes to overproduce TNF-¿ by about 4-fold. Further, the same supernatant increased CCL2 and CXCL8 production in endothelial cells. We used RNA microarray to query RNA regulation in the macaque endothelium caused by SIVmac251 infection. These studies confirm that endothelial inflammation/activation is driven by pro-inflammatory cytokines, especially TNF-¿. The activation of the TNF-¿ receptor in endothelial cells by its ligand causes activation of NF¿B in the endothelium. This leads to the subsequent upregulation/phosphorylation of MLCK, actin cytoskeletal reorganization, and redistribution of ZO-1. Displacement of ZO-1 leads to dysfunction of the endothelial tight junction possibly mediated through JAM-2 and claudin 8. We were able to largely mitigate changes in SIVmac251-driven mRNA changes in primary brain microvascular endothelial cells with inhibitors specific to MLCK and FAK.