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。在我们的体内血管损伤的新模型中，在TNF-24.0小时，有一个“窗口”的抑制血管通透性在TNF-4.0小时。我们的新的初步数据显示，在体外和/或在体内，抑制（~4.0小时）TNF诱导的内皮细胞通透性增加与核-连环蛋白易位和增加总的和膜VE-钙粘蛋白。我们的新的初步数据表明：（1）TNF-24小时诱导的屏障功能障碍离体抑制糖原合成酶激酶（GSK）3a/<$在体内，（2）T细胞因子（TCF）/淋巴增强因子（LEF）/<$- 连环蛋白依赖性启动子活性在体外和体内被TNF增加，（3）在体内抑制PKC和在体外抑制PKCa可阻止TNF对这些反应。本提案将测试TNF诱导的（TNF后约24.0小时）血管通透性增加至少部分受到PKCa诱导的GSK 3？活性抑制（TNF后约4.0小时）的抑制的范例。GSK 3活性的降低导致核连环蛋白的增加。基因组的连环蛋白活性增加VE-钙粘蛋白的表达。VE-钙粘蛋白表达的增加促进小带粘附连接抑制（即，制动）的初始（约0.5小时后TNF）增加内皮蛋白渗透性。VE-钙粘蛋白的增加促进了与<$-连环蛋白的复合物形成，这是<$-连环蛋白连续基因组效应的关闭信号。建议持续抑制GSK 3活性将继续抑制屏障功能障碍。我们将绘制一个新的范例（即，体外、原位和体内）用于抑制（“制动”）TNF诱导的肺损伤，所述肺损伤是GSK 3介导的、依赖于β-连环蛋白的VE-钙粘蛋白活性增加。假设：假设要测试的是肿瘤坏死因子诱导的肺损伤被抑制，至少部分，？PKCa？? GSK3？? 2-连环蛋白？?粘附连接中的VE-钙粘蛋白。具体目的是在体外和/或体内确定内皮细胞中：（1）TNF引起PKCa介导的GSK 3 <$-抑制，从而诱导核<$-连环蛋白易位（1- 2年），（2）TNF诱导的核-连环蛋白易位导致VE-钙粘蛋白增加：（I）启动子活性，（ii）RNA表达，和（iii）蛋白质合成（2-3年），和（3）VE-钙粘蛋白表达的增加至少部分抑制，后者TNF诱导的血管屏障功能障碍增加（3-4）。该提议将使用大鼠肺微血管内皮和从体内治疗的大鼠分离的肺。一系列生化、基因组、蛋白质组和细胞生物学测定与肺部生理学结果相结合。这种方法将在体内将细胞-分子途径转化为肺损伤的机制。

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.