Sphingosine 1-phosphate and PMN Ca2+ entry in trauma

说明(申请人提供)：创伤是现代武装冲突以及几乎每一次恐怖袭击中死亡和残疾的主要原因。不太为人所知的是，创伤是45岁以下平民死亡的最常见原因。在出生到36岁之间，创伤超过了所有其他死因的总和。许多患者最初在受伤后存活，但在创伤和休克引发中性粒细胞(PMN)炎症和功能障碍导致器官衰竭或脓毒症时，却遭受长期的危重疾病或死亡。因此，休克后PMN介导的炎症反应是一个重要的国防和公共卫生问题。在我们上一次的赠款支持期间，我们发现创伤后的炎症反应了环境中钙离子进入PMN的异常调节。这些途径在一定程度上依赖于激动剂启动的细胞钙库排空，因此通常被称为钙库操作的钙内流(SOCE)。我们进一步证明，在PMN中，SOCE是通过细胞合成脂质第二信使1-磷酸鞘氨醇(S1P)来响应钙库排空而介导的。相关脂类的作用类似，无论产生在细胞内还是细胞外，都能刺激PMN的激活。我们还证明了PMN SOCE是通过一个复杂的钙离子进入通道系统发生的，该通道由“瞬时受体电位”通道蛋白(TRPC)组成。由于SOCE是PMN钙离子的关键调节因子，损伤后PMN对SOCE的调节异常，这些发现提示S1P合成和SOCE特异性钙通道的药理调节可能预防PMN介导的休克和创伤的炎性后遗症。进一步的研究表明，在模拟临床相关创伤和休克治疗情景的复杂动物模型中，通过各种策略抑制SOCE可以减少PMN介导的炎症和肺损伤。本提案旨在将我们之前的工作从我们先前的基本分子生物学观察的临床应用前景阶段扩展到为战场和平民创伤实践的临床实施做好准备的众所周知的治疗策略阶段。我们建议通过实现以下四个具体目标来实现这一点：1.确定如何使用SOCE抑制来预防创伤性休克后的器官损伤2.确定SOCE抑制在休克后的副作用和并发症。3.确定创伤对细胞内S1P/SOCE信号的影响4.确定创伤后循环中S1P激活PMN的机制。

DESCRIPTION (provided by applicant): Trauma is the major cause of death and disability in modern armed conflict as well as in almost every terrorist attack. Less well known, trauma is the most common cause of civilian death below age 45. Between birth and age 36, trauma exceeds all other causes of death combined. Many patients survive injury initially only to suffer prolonged critical illness or die later when trauma and shock trigger neutrophil (PMN) inflammation and dysfunction that leads to organ failure or sepsis. Thus PMN-mediated inflammation after shock is a key national defense as well as public health problem. During our last grant support period, we showed that inflammation after trauma reflects aberrant regulation of calcium ion (Ca2+) entry from the environment into PMN. These pathways depend in part on the agonist-initiated emptying of cell Ca2+ stores and are therefore generally termed store-operated calcium entry (SOCE). We further showed that in PMN, SOCE is mediated by cellular synthesis of a lipid second messenger, sphingosine 1-phosphate (S1P) in response to Ca2+ store emptying. Related lipids act similarly, and can stimulate PMN activation whether produced intra- or extra-cellular. We have also demonstrated that PMN SOCE occurs through a complex system of Ca2+ entry channels which are composed of "Transient Receptor Potential" channel proteins (TRPC). Since SOCE is a key regulator of PMN Ca2+ and is abnormally regulated in PMN after injury, these findings suggested the hypothesis that pharmacologic modulation of both S1P synthesis and SOCE-specific Ca2+ channels might prevent PMN-mediated inflammatory sequellae of shock and trauma. Further work has now shown that inhibition of SOCE by a variety of strategies can act to diminish PMN-mediated inflammation and lung injury in complex animal models mimicking clinically relevant trauma and shock treatment scenarios. The present proposal seeks to extend our prior work from the stage of a clinical promising application of our prior basic molecular biologic observations, to the stage of a well-understood treatment strategy ready for clinical implementation both on the battlefield and in civilian trauma practice. We propose to do this by achieving the four following Specific Aims: 1. Determine how to use SOCE inhibition to prevent organ injury after traumatic shock 2. Determine the side effects and complications of SOCE inhibition after shock. 3. Determine the effects of injury on intracellular S1P/SOCE signaling 4. Determine the mechanisms by which circulating S1P activates PMN after trauma.