A mechanism for suppression of TNF induced endothelial dysfunction

描述（由申请人提供）：在肺微血管内皮细胞中，TNF（即早期~0.5小时）诱导ONOO介导的-肌动蛋白硝化，导致-连环蛋白从粘附连接处脱位。在我们新的体内血管损伤模型中，在TNF-24.0小时内存在抑制血管通透性的“窗口”。我们新的初步数据显示，在体外和/或体内，抑制（~4.0小时）tnf诱导的内皮通透性增加与核¿-catenin易位和总VE-cadherin和膜VE-cadherin的增加有关。我们新的初步数据表明：(1)体内抑制糖原合成酶激酶（GSK）3a/¿可抑制TNF-24小时诱导的体外屏障功能障碍，(2)体外和体内TNF可增加T细胞因子(TCF)/淋巴细胞增强因子(LEF)/ -catenin依赖性启动子活性，(3)体内和体外抑制PKC可阻止这些对TNF的反应。这一提议将验证TNF诱导的血管通透性增加（TNF-后~24.0小时）至少部分被pkca诱导的GSK3¿活性抑制（TNF-后~4.0小时）所抑制的范式。GSK3活性的降低导致核连环蛋白的增加。基因组的¿-catenin-活性增加了VE-cadherin的表达。VE-cadherin表达的增加促进了结节粘附连接的抑制（即抑制）初始（tnf后约0.5小时）内皮蛋白通透性的增加。ve -钙粘蛋白的增加促进了与-catenin的复合物形成，这是-catenin连续基因组效应的关闭信号。研究人员提出，持续抑制GSK3¿活性将继续抑制屏障功能障碍。我们将绘制一种新的范式（即体外、原位和体内）来抑制（“制动”）tnf诱导的肺损伤，这是GSK3介导的、依赖于catenin的ve -钙粘蛋白活性的增加。假设：待验证的假设是tnf诱导的肺损伤被抑制，至少部分地被？PKCa吗?？GSK3害怕吗?？2-catenin吗?？粘附连接中的ve -钙粘蛋白。具体目的是在体外和/或体内内皮中确定：(1)TNF引起pkca介导的GSK3 -抑制，诱导核catenin易位（1 - 2年）；(2)TNF诱导的核catenin易位导致VE-cadherin增加；(I)启动子活性，（ii） RNA表达，（iii）蛋白质合成（第2-3年），(3)VE-cadherin蛋白表达的增加至少部分抑制了后者tnf诱导的血管屏障功能障碍的增加（3-4）。本研究拟采用大鼠肺微血管内皮和离体大鼠肺。一系列生化、基因组、蛋白质组学和细胞生物学检测与肺生理学结果相结合。这种方法将细胞分子途径转化为体内肺损伤的机制。

DESCRIPTION (provided by applicant): In lung microvessel endothelial cells, TNF (i.e., early ~0.5 hr) induces ONOO--mediated nitration of ¿-actin causing dislocation of ¿-catenin from adherence junctions. In our new model of in vivo vascular injury at TNF-24.0 hr, there is a "window" of suppressed vascular permeability within TNF-4.0 hr. Our new preliminary data shows, both in vitro and/or in vivo, that the suppression of (~4.0 hr) TNF-induced increased endothelial permeability is associated with nuclear ¿-catenin translocation and increases in total and membrane VE-cadherin. Our novel preliminary data demonstrates: (1) TNF-24 hr induced barrier dysfunction ex vivo is suppressed by inhibition of glycogen synthetase kinase (GSK)3a/¿ in vivo, (2) T cell factor (TCF)/ lymphoid enhancer factor (LEF)/¿-catenin dependent promoter activity is increased by TNF in vitro and in vivo, and (3) These responses to TNF are prevented by inhibition of PKC in vivo and PKCa in vitro. This proposal will test the paradigm that TNF-induced (~24.0 hr post- TNF) increase in vascular permeability is suppressed (~4.0 hr post-TNF), at least in part, by PKCa-induced inhibition of GSK3¿ activity. The decrease in GSK3¿ activity causes increased nuclear ¿-catenin. The genomic ¿-catenin-activity increases the expression of VE-cadherin. The increase in VE-cadherin expression promotes zonular-adherence junctions suppressing (i.e., braking) the initial (~0.5 hr post-TNF) increased endothelial protein permeability. The increase in VE-cadherin promotes complex formation with ¿-catenin, the off signal for continuous genomic effects of ¿-catenin. It is proposed that persistent inhibition of GSK3¿ activity will continue to suppress barrier dysfunction. We will map a novel paradigm (i.e., in vitro, in situ and in vivo) for the suppression ("braking") of TNF-induced lung injury which is GSK3¿-mediated, ¿-catenin-dependent increased VE-cadherin activity. Hypothesis: The hypothesis to be tested is TNF-induced lung injury is suppressed, at least in part by, ?PKCa ? ?GSK3¿ ? ?2-catenin ? ?VE-cadherin in adherence junctions. The Specific Aims are to determine, in vitro and/or in vivo, in endothelium that: (1) TNF causes PKCa-mediated GSK3¿-inhibition that induces nuclear ¿-catenin translocation (Years 1- 2), (2) The TNF-induced nuclear ¿-catenin translocation causes increased VE-cadherin: (I) promoter activity, (ii) RNA expression, and (iii) protein synthesis (Year 2-3), and (3) The increase in VE-cadherin protein expression suppresses, at least in part, the latter TNF-induced increase in vascular barrier dysfunction (3-4). This proposal will use rat lung microvessel endothelium and lungs isolated from rats treated in vivo. An array of biochemical, genomic, proteonomic and cell biology assays are integrated with lung physiology outcomes. This approach will translate cell-molecular pathways to the mechanisms of lung injury in vivo.