Modulation eNOS Coupling In Gram Positive Infection-Associated Acute Lung Injury

项目总结(见说明)： 革兰氏阳性感染约占所有急性肺损伤病例的50%，肺炎链球菌感染占所有社区获得性肺炎(CAP)病例的45%。CAP伴有广泛的通透性水肿，其特征是内皮屏障完整性的破坏。CAP严重程度的一个主要因素是细菌毒力因子的分泌，主要是肺炎溶血素(PLY)和家族成员李斯特溶素-0(LLO)。这些革兰氏阳性毒力因子使质膜毛孔引起不同类型的细胞内钙内流，刺激宿主PLC活性，并激活蛋白激酶Cα(PKCalpha)。此前，我们已经证明，对PKCalpha的抑制对这些革兰氏阳性毒力因子具有屏障保护作用。此外，这至少部分归因于PKCα增加相关的活性氧和氮物种的产生的能力。LIO诱导eNOS解偶联，增强过氧亚硝酸根的生成，增加RhoA和Rac-1硝化，从而激活前者，抑制后者。本项目将侧重于eNOS解偶联的不同机制，我们假设eNOS解偶联是通过PKCalpha介导的eNOS在Thr495的磷酸化发生的，而不是通过增加ADMA产生(见项目1)。根据我们之前发表的研究和新的初步数据，我们将在本提案中检验的总体假设是，抑制PKCalpha导致eNOS释放NO增加，从而刺激RhoA和Rac1的S亚硝化。 这种修饰导致RhoA抑制、rac1激活和EC屏障保护。具体目标1将确定PKCalpha介导PLY/LLO驱动的eNOS解偶联的机制。具体目标2将确定PKCalpha介导的NO信号增加是否减轻PLY和LLO诱导的内皮屏障破坏，以及这是否通过RhoA和rac1的S亚硝化发生。这一目标还将确定RhoA和rac1上被亚硝化的特定细胞因子残基。特异靶3将在体内确定降低PKCalpha活性和直接增强RhoA和rac1亚硝化在保护ALI时内皮屏障中的相对作用。

PROJECT SUMMARY (See instructions): Gram positive infections make up ~50% of all acute lung injury cases with Streptococcus pneumonia infections accounting for 45% of all community-acquired pneumonia (CAP) cases. CAP is accompanied by extensive permeability edema, characterized by a disruption in endothelial barrier integrity. A major factor in the severity of CAP is the secretion of bacterial virulence factors predominantly pneumolysin (PLY) and family member listeriolysin-0 (LLO). These gram positive virulence factors make plasma membrane pores that cause a Ca2+-influx in various cell types, stimulate host PLC activity and as we have recently shown, activate protein kinase C alpha (PKCalpha). Previously we showed that inhibition of PKCalpha is barrier protective against these gram-positive virulence factors. Further, this is due, at least in part, to the ability of PKC alpha to increase production of reactive oxygen- and -nitrogen species associated. LLO induces eNOS uncoupling, enhances peroxynitrite generation, and causes an increase in RhoA and Rac 1 nitration resulting in activation of the former and inhibition of the latter. This project will focus on distinct mechanisms of eNOS uncoupling that we hypothesize occurs via the PKCalpha mediated phosphorylation of eNOS at Thr495 rather than through increased ADMA generation (see Project 1). From our previously published studies and new preliminary data, the overall hypothesis that we will test in this proposal is that inhibition of PKCalpha leads to the enhanced release of NO from eNOS which stimulates the S-nitrosylation of RhoA and Rac 1. This modification leads to RhoA inhibition and Rac1 activation and EC barrier protection. Specific Aim 1 will determine the mechanism by which PKCalpha mediates PLY/LLO driven eNOS uncoupling. Specific Aim 2 will determine if PKCalpha-mediated increases in NO signaling attenuate the endothelial barrier disruption induced by PLY and LLO and whether this occurs via the S-nitrosylation of RhoA and Rac1. This Aim will also identify the specific cytokine residues on RhoA and Rac1 that are nitrosylated. Specific Aim 3 will determine the relative effects of reducing PKCalpha activity and directly enhancing RhoA and Rac1 Snitrosylation in protecting the endothelial barrier during ALI in vivo.