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

项目总结（见说明）： 革兰氏阳性菌感染占所有急性肺损伤病例的约50%，肺炎链球菌感染占所有社区获得性肺炎（CAP）病例的45%。CAP伴有广泛的渗透性水肿，其特征在于内皮屏障完整性的破坏。CAP严重性的一个主要因素是细菌毒力因子的分泌，主要是肺炎球菌溶血素（Pneumolysin，Pneumolysin）和家族成员肺炎球菌溶血素-0（LLO）。这些革兰氏阳性毒力因子使质膜孔，引起各种细胞类型中的Ca 2+内流，刺激宿主PLC活性，并且如我们最近所示，激活蛋白激酶C α（PKCalpha）。以前，我们表明，PKCalpha的抑制是对这些革兰氏阳性毒力因子的屏障保护。此外，这至少部分是由于PKC α增加相关活性氧和氮物质产生的能力。LLO诱导eNOS解偶联，增强过氧亚硝酸根的产生，并导致RhoA和Rac 1硝化的增加，导致前者的激活和后者的抑制。该项目将重点关注eNOS解偶联的不同机制，我们假设通过PKCalpha介导的eNOS在Thr 495的磷酸化而不是通过增加ADMA的产生（见项目1）。根据我们先前发表的研究和新的初步数据，我们将在该提议中测试的总体假设是，PKCalpha的抑制导致NO从eNOS的释放增强，其刺激RhoA和Rac 1的S-亚硝基化。 这种修饰导致RhoA抑制和Rac 1激活和EC屏障保护。具体目标1将确定PKCalpha介导eNOS/LLO驱动的eNOS解偶联的机制。具体目标2将确定PKCa介导的NO信号传导的增加是否减弱由RhoA和LLO诱导的内皮屏障破坏，以及这是否通过RhoA和Rac 1的S-亚硝基化发生。该目的还将鉴定RhoA和Rac 1上被亚硝基化的特定细胞因子残基。具体目标3将确定降低PKCalpha活性和直接增强RhoA和Rac 1亚硝基化在体内ALI期间保护内皮屏障的相对效果。

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.