Endothelial Toll-Like Receptor-4 Signaling in Sepsis

描述（由申请人提供）： 脓毒症和脓毒症综合征是危及生命的疾病，是重症监护病房死亡的主要原因。败血症最常见的是由革兰氏阴性菌引起的。脂多糖（LPS）是细菌壁的重要组成部分，有助于脓毒症的发病机制。内皮细胞（EC）是LPS和促炎细胞因子白细胞介素-1 β（IL-1 β）和肿瘤坏死因子-α（TNF α）的主要靶点。脂多糖、IL-1 β和TNF α均直接激活EC并引发一系列EC反应，包括细胞因子、趋化因子、粘附分子和促凝血蛋白的上调。大多数这些促炎反应是由转录因子NF-κ B介导的，这对细胞存活也很重要。因此，内皮细胞活化被认为在脓毒症中宿主防御和全身炎症反应的发展中起关键作用。越来越多的证据表明，EC凋亡参与了实验性脓毒症的发病机制。事实上，已经表明活化蛋白C（唯一批准用于治疗人类严重脓毒症的药物）的有益作用的重要组成部分可能是由于其对EC的抗凋亡作用。 转导LPS信号的细胞受体已被鉴定为Toll样受体（TLR）4，TLR受体大家族的成员。在这个建议中，我们将进一步研究促炎，促生存，促凋亡信号转导的TLR 4在体外EC的分子机制，并确定EC激活和凋亡的作用，在体内使用转基因小鼠盲肠结扎穿孔（CLP）诱导的实验性脓毒症。具体目标是：1）研究磷脂酰肌醇-3激酶（phosphoinositide-3 kinase，PI 3-K）在LPS/TLR 4刺激EC存活中的作用，2）研究LPS/TLR 4诱导的EC促凋亡信号通路的机制，3）鉴定TLR 4诱导的TRAF 6信号复合物的新组分，4）研究LPS/TLR 4诱导的TRAF 6信号复合物在EC存活中的作用。（4）探讨诱导性、选择性阻断EC NF-κ B活化和凋亡对CLP诱导脓毒症的影响。

DESCRIPTION (provided by applicant): Sepsis and sepsis syndromes are life-threatening conditions and are leading causes of death in intensive care units. Sepsis is most commonly caused by Gram-negative bacteria. Lipopolysaccharide (LPS) is an important component of the bacteria wall that contributes to the pathogenesis of sepsis. Endothelial cells (ECs) are a major target of LPS and the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha). Lipopolysaccharide, IL- 1beta, and TNFalpha all directly activate ECs and elicit an array of EC responses, including the upregulation of cytokines, chemokines, adhesion molecules, and procoagulant proteins. Most of these pro-inflammatory responses are mediated by the transcription factor NF-KappaB, which is also important for cell survival. Endothelial cell activation is thus thought to play a critical role in the development of both host defense and the systemic inflammatory response in sepsis. Increasing evidence also indicates that EC apoptosis contributes to the pathogenesis of experimental sepsis. Indeed, it has been suggested that a significant component of the beneficial effects of activated protein C, the only approved drug for the treatment of severe sepsis in man, may be due to its anti-apoptotic effects on ECs . The cellular receptor transducing the LPS signal has been identified as Toll-like receptor(TLR)4, a member of the larger family of TLR receptors. In this proposal we will examine further the molecular mechanisms of pro-inflammatory, pro-survival, and pro-apoptotic signaling by TLR4 in EC in vitro and determine the role of EC activation and apoptosis in vivo using transgenic mice in experimental sepsis induced by cecal ligation and puncture (CLP). The specific aims are: 1) To determine the role of phosphoinositide-3 kinase (PI3-K) in EC survival following LPS/TLR4 stimulation; 2) To determine the mechanisms of LPS/TLR4-lnduced pro-apoptotic signaling in ECs; 3) To identify new components of the TLR4-induced TRAF6-signaling complex in ECs; and 4) To determine the effect of inducible, selective blockade of EC NF-KappaB activation and apoptosis on sepsis induced by CLP.