In-vivo and Ex-vivo Analysis of Tissue S-nitrosothiols in Sepsis

About This Grant

PROJECT SUMMARY Each year there is an estimated eight million deaths globally that are attributed to sepsis. Sepsis is one of the leading causes of death in the pediatric population. When children die it is often from refractory shock and development of multi-organ failure. A key driver of this refractory shock is vascular dysfunction. Nitrosative stress has been linked to the pathophysiology of sepsis and is associated with this vascular dysfunction. However, the role of nitrogen oxides is incompletely understood in sepsis. There is a need to define the role of nitrogen oxides to identify new and effective therapeutic targets. S-nitrosothiols are endogenous nitrogen oxides that are exquisitely bioactive in vascular physiology. Many circulating S-nitrosylated proteins have established physiological roles. In addition, certain low-mass S-nitrosothiols have important physiologic roles and have been recently demonstrated to be stored and released from extracellular vesicles (EVs). In adults S-nitrosothiol levels are high when measured ex-vivo in sepsis. Unfortunately, due to their lability, measurement can be very difficult, thus limiting clinical utility. Our team has recently designed and validated a device that allows for non-invasive measurement of tissue S-nitrosothiols in-vivo. Our objective is to define the role of S-nitrosothiols in blood and EVs and to establish their relationships in sepsis. Our hypothesis is that circulating concentrations of S- nitrosothiols, including those in EVs, are increased in sepsis and that these high levels are associated with worse outcomes. My long-term career goal is to develop a research portfolio studying the biology of these molecules and to leverage this understanding to develop new therapies for sepsis. This K08 proposal summarizes a 5-year program of mentored support tied to a research project to better determine the roles of S-nitrosylation signaling in sepsis. Our specific aims for this project are to: (1) test the hypothesis that EV cargo is enriched with S- nitrosothiols in a murine model of sepsis and to identify novel S-nitrosylated proteins associated with sepsis- related pathways and (2) test the hypothesis that S-nitrosothiols are elevated in EV cargo from pediatric patients with sepsis, and their levels correlate with device-based measurements and predict clinical outcomes. Through my career development plan and ongoing support and education from my mentors, I will develop expertise in a) bioinformatics and proteomics, b) mouse husbandry, and c) EV biology and research techniques. Together the research and career development aims of this proposal will provide me with the necessary skills and education to compete for additional funding as an independent investigator. Specifically, I plan to submit for an R01 application through the NHLBI in year 5 of this career development award period. This proposal will focus on investigating the mechanistic underpinnings for the differentially expressed S-nitrosothiols contained in EVs. Overall, this proposal is an important first step in establishing my career as an independent investigator with a focus on pediatric sepsis.