Erythrocyte Nitrosothiol Flux and Vasoregulation in Lung

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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.