Erythrocyte Nitrosothiol Flux and Vasoregulation in Lung

描述(由申请者提供)：本建议书描述了一项五年计划，旨在获得重症监护领域学术内科医生-科学家职业发展所需的研究技能。应聘者是儿科重症监护科的初级教员，接受过肺生理学的初步研究培训。该奖项将被用来拓宽这一背景，获得血管细胞生物学和亚硝化信号生物化学的基础，以便能够独立研究血管运动控制中断的病理生物学中的红细胞和内皮通讯。肺亚硝硫醇信号研究的领先者、医学博士Ben Gaston将担任该奖项的主要导师，微循环研究和血管细胞生物学的先驱Brian Duling博士将担任共同导师。亚硝化血红蛋白化学方面的专门知识将通过与杜克大学和宾夕法尼亚大学的研究人员合作提供。此外，一个由资深科学家组成的咨询委员会将提供科学和职业指导。与该奖项进行的研究将通过一氧化氮(NO)和血红蛋白(Hb)的相互作用来调查红细胞作为肺血流失调和远程炎症之间的联系所起的作用。有证据表明，在亚硝化信号网络中，Hb和NO的反应是平衡的，以传递局部氧化还原梯度，耦合氧分压和NO在血管床中的分布(从而导致流动)。在这一点上，我们假设全身炎症反应综合征(SIRS)中的氧化应激可能扰乱正常的红细胞亚硝化信号，并解释这种状态下肺血流异常的原因。在这个项目中，我们的目标是确定(1)SIRS中红细胞NO负荷异常的程度，与呼吸衰竭的发生和严重程度相关，(2)确定在β-链半胱氨酸(β-Chain cysteine，β-半胱氨酸)中加入NO后肺内Hb血管活性的变化，这种变化的变构控制，以及(3)基于Hb的亚硝化信号的内皮调节。我们将在三个层面上追求这些目标：(1)几乎没有化学成分的红细胞内Hb的分子研究(2)培养中红细胞和内皮细胞之间NO信号的药理学、免疫组织化学和蛋白质组学研究，以及(3)我们在分离的小鼠肺中的发现的生理学相关性，从而允许进一步的转基因和药理学查询。在本项目结束时，我们期望通过一系列的反硝化反应来确定远端血管床和肺之间通过红细胞的NO交通的机制。在发育阶段结束时，候选人将掌握进一步独立研究(S)一氧化氮和红细胞血管活性的技能，希望为针对肺局部血流调节失调的治疗提供信息。候选人的长期目标是确定通过红细胞在区域血管床之间没有交通的机制，以及它在严重炎症状态下多器官衰竭演变中的作用。

DESCRIPTION (provided by applicant): This proposal describes a five-year plan for the attainment of research skills necessary in the career development of an academic physician-scientist in Critical Care. The candidate is a junior faculty member in an established Division of Pediatric Critical Care and has preliminary research training in pulmonary physiology. The award will be utilized to broaden this background by acquiring a foundation in vascular cell biology and the biochemistry of nitrosative signaling to enable independent investigation of erythrocytic and endothelial communication in the pathobiology of disrupted vasomotor control. Ben Gaston, MD, a leader in pulmonary nitrosothiol signaling research, will serve as primary mentor for the award and Brian Duling, PhD, a pioneer in microcirculation research and vascular cell biology will serve as Co-Mentor. Specific expertise in nitrosative hemoglobin chemistry will be provided by collaboration with investigators from Duke and the University of Pennsylvania. Additionally, an advisory committee of senior scientists will provide scientific and career guidance. Research conducted with this award will investigate the role of the erythrocyte as a link between dysregulated pulmonary blood flow and remote inflammation via nitric oxide (NO) and hemoglobin (Hb) interactions. There is evidence for a nitrosative signaling network in which Hb and NO reactions are balanced to transduce regional redox gradients, coupling oxygen tension and the distribution of NO (and thus flow), in vascular beds. In this regard, we hypothesize oxidative stress in the systemic inflammatory response syndrome (SIRS) may disrupt normal erythrocytic nitrosative signaling and explain dysregulated pulmonary blood flow in this state. We aim, in this project, to determine (1) the degree of abnormal NO loading of RBCs in SIRS, correlating with onset and severity of respiratory failure and (2) to determine the change in Hb vasoactivity in the lung following addition of NO to a beta-chain cysteine (betacys93), the allosteric control of this change, and (3) the endothelial regulation of Hb-based nitrosative signaling. We will pursue these goals on three levels: (1) molecular investigation of intraerythrocytic Hb approximately NO chemistry (2) pharmacologic, immunohistochemical, and proteomic investigations of NO signaling between erythrocytes and endothelial cells in culture, and (3) physiologic correlation of our findings in the isolated murine lung, permitting further transgenic and pharmacologic query. At the conclusion of this project, we expect to define the mechanism of NO traffic between remote vascular beds and the lung via the erythrocyte, as effected by serial transnitrosation reactions. At the conclusion of the development period, the candidate will have acquired skills to pursue further independent investigation of (S)NO and erythrocyte vasoactivity with the hope of informing therapy directed at the dysregulation of regional blood flow in the lung. The candidate's long-term goal is to define the mechanism of NO traffic between regional vascular beds via the erythrocyte, and its role in the evolution of multiple organ failure in severe inflammatory states.