Endothelial Toll-Like Receptor-4 Signaling in Sepsis

描述(由申请人提供)： 败血症和败血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。败血症最常见的原因是革兰氏阴性菌。脂多糖是细菌壁的重要组成部分，参与了脓毒症的发病机制。内皮细胞(ECs)是内毒素和促炎细胞因子白介素1β(IL 1β)和肿瘤坏死因子α(TNFpha)的主要靶点。脂多糖、IL-1β和肿瘤坏死因子α都能直接激活内皮细胞，引起一系列的内皮细胞反应，包括细胞因子、趋化因子、黏附分子和促凝血蛋白的上调。这些促炎反应中的大多数是由转录因子NF-kappaB介导的，这对细胞的生存也很重要。因此，内皮细胞的激活被认为在脓毒症的宿主防御和全身炎症反应的发展中起着关键作用。越来越多的证据表明EC细胞凋亡在实验性脓毒症的发病机制中起重要作用。事实上，有研究表明，活性蛋白C的有益作用的一个重要组成部分可能是由于其对内皮细胞的抗凋亡作用。激活蛋白C是唯一被批准用于治疗人类严重脓毒症的药物。 转导内毒素信号的细胞受体被鉴定为Toll样受体(TLR)4，是TLR受体大家族中的一员。在这项研究中，我们将在体外进一步研究TLR4在EC中促炎、促存活和促凋亡信号的分子机制，并利用转基因小鼠在盲肠结扎和穿孔(CLP)诱导的实验性脓毒症中确定EC的激活和凋亡在体内的作用。其具体目的是：1)确定磷脂酰肌醇-3激酶(PI3-K)在脂多糖/TLR4刺激后内皮细胞存活中的作用；2)确定内毒素/TLR4诱导内皮细胞凋亡信号的机制；3)鉴定TLR4诱导内皮细胞TRAF6信号复合体的新成分；以及4)确定可诱导的、选择性地阻断EC-NF-kappaB激活和凋亡对CLP诱导的脓毒症的影响。

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.