Effects of Angiopoietins on Shock-Induced Acute Lung Injury

项目总结(见说明)： 内皮细胞屏障功能的丧失在间接急性肺损伤(ALI)的发生发展中起重要作用。在一种新的小鼠出血(休克)模型中，盲肠结扎和改形(CLP)导致随后的败血症挑战，中性粒细胞(PMN)与常驻肺细胞的相互作用是这种病理的中心。在ALI中，未解决的炎症导致EC屏障完整性丧失和肺功能受损的病理过程。在生理条件下，内皮细胞生长因子血管生成素(Ang)-I和血管生成素-2(Ang-1)和血管生成素-2(Ang-2)通过与内皮细胞上表达的T3T：Osine Kinase Receptor(Tie2)竞争性相互作用维持血管内环境的稳定。Ang-1/Tie2结合被证明能稳定血管并刺激下游促生存/抗炎信号，相反，Ang-2从激活的内皮细胞储存颗粒中释放出来，破坏血管的稳定。最近的研究发现，发生ALI的患者血浆Ang-2水平显著升高。在我们的休克/CLP模型中，我们在肺和血浆中发现了类似的升高，我们发现休克前PMN的耗尽会抑制Ang-2的升高。我们提出以下中心假设：血管紧张素转换酶-2导致休克/CLP所致的ALI肺内皮细胞屏障功能丧失，这是由EC与休克启动的PMN相互作用所引发的。我们提出以下具体目标：目标1将确定Ang-1的变化动力学：Ang-2和Ang-2的表达和再合成，以及它与炎症指标变化的关系。我们将利用Ang-2(Si)RNA抑制肺组织表达、Ang-2蛋白特异性抑制和Ang-1竞争性抑制Ang-2/Tie2结合，以评估休克预适应中Ang-2释放在ALI发生发展中的作用。目的研究血管紧张素转换酶2(Ang-2)改变ALI/ARDS小鼠血浆肺内皮细胞表型/活化的机制。目的研究血管紧张素转换酶-2(Ang-2)介导休克诱导的中性粒细胞与培养的小鼠内皮细胞共培养后内皮细胞表型/活化改变的机制。该方案中的研究将为PMN相关的Ang-2介导的ALI的机制提供新的见解，并将阐明潜在的治疗干预途径。

PROJECT SUMMARY (See instructions): Loss of endothelial cell (EC) barrier function is important in the development of indirect acute lung injury (ALI). We have shown, in a novel murine model of hemorrhage (shock) with a subsequent septic challenge caused by cecal ligation and performation (CLP), that neutrophil (PMN) interactions with resident pulmonary cells are central to this pathology. In ALI, unresolved inflammation elicits a pathological process with loss of EC barrier integrity and impaired lung function. EC growth factors, Angiopoietin (Ang)-i and 2, under physiological conditions, maintain vascular homeostasis through competitive interactions wdth the t3T:osine kinase receptor, Tie2, expressed on ECs. Ang-1/Tie2 binding has been shown to stabilize vessels and stimulate down stream pro-survival/anti-inflammatory signaling, in contrast, Ang-2, released from storage granules of activated ECs, destabilizes vessels. Recent findings report that plasma Ang-2 levels are significantly elevated in patients that develop ALI. We find similar elevation in the lungs and plasma in our shock/CLP model, and we have found that depletion of PMNs prior to shock abrogates Ang-2 elevation. We propose the following central hypothesis: Ang-2 causes loss of pulmonary EC barrier function in ALI due to shock/CLP, initiated by EC interaction with shock-primed PMNs. We propose the following specific aims: Aim 1 will determine the kinetics of change in Ang-1:Ang-2 and Ang-2 expression and re-synthesis as well as its relationship to changes in indices of inflammation. We will use Ang-2 (si)RNA to suppress lung tissue expression, Ang-2 protein specific inhibition, and Ang-1 competitive inhibition of Ang-2/Tie2 binding to assess the contribution of Ang-2 release in shock priming for the development of ALI. Aim 2 will determine mechanisms by which Ang-2 changes pulmonary EC phenotype/activation in response to plasma from mice with ALI/ARDS. Aim 3 will determine mechanisms by which Ang-2 mediates changes in EC phenotype /activation in cultured mouse ECs following co-culture with shock-primed PMNs. The studies in this proposal will provide novel insights into the mechanisms of PMN associated, Ang-2 mediated ALI and will elucidate pathways that hold potential for therapeutic intervention.