Inhibition of the Fas/FasL system in experimental acute lung injury

 描述（由申请人提供） 急性呼吸窘迫综合征（ARDS）在美国是一个重要的临床问题，每年影响200，000名患者，并导致约75，000人死亡。本实验室对Fas/FasL系统在人类和动物急性肺损伤（ALI）病理生理学中的作用感兴趣。该系统由膜表面受体Fas（CD 95）及其同源配体FasL（CD 178）组成。Fas与FasL的结合激活导致细胞凋亡和细胞因子释放的信号传导途径。我们已经表明，激活Fas/FasL系统的动物导致ALI，而小鼠缺乏功能性Fas的保护LPS诱导的肺损伤。我们还表明，Fas/FasL系统在患有ARDS的人的肺中是活跃的。最近我们发现sFasL在肺中的生物学活性依赖于其结构。具体来说，我们确定sFasL存在于至少两种形式：一个144个氨基酸的短形式，主要由结合域，和一个178个氨基酸的长形式，由结合域加上一个短的质膜“茎”区。只有sFasL的长型，这是存在于患有ARDS的患者的BAL液中的主要形式。重要的是，我们已经发现，其中茎区的所有8个带电荷的氨基酸已被改变为丙氨酸的sFasL突变体具有降低的功能，并且此外，在体外充当天然sFasL的抑制剂。我们现在试图确定8位点突变的sFasL的抑制机制以及它是否能减轻体内肺损伤。假设：8位点突变的sFasL通过损害Fas受体形成启动信号传导所需的膜簇的能力而充当天然sFasL的抑制剂。因此，8位点突变的sFasL将减弱体外人ARDS BALF的促凋亡活性和体内实验性急性肺损伤的严重程度。目标1：确定8位点突变的sFasL是否通过损害Fas：FasL簇的形成和随后的受体：配体加帽来抑制天然sFasL。目的2：确定8位点突变的sFasL是否在体外减弱来自患有ARDS的人的BALF的促凋亡活性。目的3：确定8位突变的sFasL是否减轻实验性急性肺损伤的严重程度。这项研究的发现将通过重新关注促凋亡系统在急性肺损伤中的机制作用以及发现新的潜在的ARDS治疗药物，在该领域产生变革性影响。

 DESCRIPTION (provided by applicant) The acute respiratory distress syndrome (ARDS) is an important clinical problem in the United States, affecting 200,000 patients per year and resulting in death of approximately 75,000 persons. Our laboratory is interested in the role of the Fas/FasL system in the pathophysiology of acute lung injury (ALI) in humans and animal models. This system is comprised of the membrane surface receptor Fas (CD95) and its cognate ligand, FasL (CD178). Binding of Fas to FasL activates signaling pathways that lead to apoptosis and also to cytokine release. We have shown that activation of the Fas/FasL system in animals leads to ALI, whereas mice lacking functional Fas are protected in LPS-induced lung injury. We have also shown that the Fas/FasL system is active in the lungs of humans with ARDS. Recently we have found that the biological activity of sFasL in the lungs is dependent on its structure. Specifically, we determine that sFasL exists in at least two forms: a 144- amino acid short form, consisting primarily of the binding domain, and a 178 amino-acid long form, consisting of the binding domain plus a short juxtamembrane "stalk" region. Only the long form of sFasL, which is the major form that is present in BAL fluid from patients with ARDS. Importantly, we have found that a sFasL mutant in which all 8 charged amino acids of the stalk region have been changed to alanines has decreased function and furthermore, acts as an inhibitor of native sFasL in vitro. We now seek to determine the mechanism of inhibition of the 8-site mutated sFasL and whether it attenuates lung injury in vivo. Hypothesis: The 8-site mutated sFasL acts as an inhibitor of native sFasL by impairing the ability of the Fas receptor to form membrane clusters that are needed to initiate signaling. Therefore, the 8-site mutated sFasL will attenuate the pro-apoptotic activity of human ARDS BALF in vitro and the severity of experimental acute lung injury in vivo. Aim 1: Determine whether the 8-site mutated sFasL inhibits native sFasL by impairing the formation of Fas:FasL clusters and subsequent receptor:ligand capping. Aim 2: Determine whether the 8-site mutated sFasL attenuates the pro-apoptotic activity of BALF from humans with ARDS in vitro. Aim 3: Determine whether the 8-site mutated sFasL attenuates the severity of experimental acute lung injury in vivo. Findings derived from this study will have a transformative impact in the field by refocusing attention on the mechanistic role of pro-apoptotic systems in acute lung injury, and by discovering new potential therapeutic agents for ARDS.