Blood transfusions are the most common therapy for severe anemia or blood loss, and offer undeniable life- saving benefits in a variety of clinical situations. Nevertheless, chronic shortages of donor blood and continued risks of infectious and immune complications (greatly reduced but not eliminated by screening) post- transfusion have spurred a search for alternatives to whole blood. Purified hemoglobin, and the hemoglobin- based oxygen carriers (HBOCs) that have been derived from it, are one potential substitute for blood transfusions. In clinical studies, however, HBOCs have consistently demonstrated adverse effects, including severe hypertension, renal injury and failure, and other end-organ damage. Extravasation and nitric oxide (NO) scavenging by the protein is thought to underlie these effects. Our lab has developed recombinant mutant proteins based on the hexacoordinate globins neuroglobin (rNgb) and cytoglobin (rCgb) that rapidly generate NO via reduction of nitrite, potentially offsetting any NO scavenging. We, therefore, propose the hypothesis that specific mutations of rNgb and rCgb can be used to engineer a new generation of oxygen carriers that cause reduced hypertension and renal toxicity. To test the hypothesis, we will use UV-Vis spectroscopy to characterize the function of numerous rNgb and rCgb mutants, and identify those mutants with ideal characteristics for oxygen transport and NO generation. We will also encapsulate these proteins within artificial lipid membranes, creating protein-containing liposomes that will be unable to extravasate. Second, we will evaluate cytochrome b5, which reduces methemoglobin in vivo, as a potential reducing system for these mutant proteins, as persistent protein oxidation prevents oxygen binding and nitrite reduction to NO. Finally, we will create liposomes (as in aim 1) containing a promising protein for oxygen delivery as well as the reducing system evaluated in aim 2, and administer these liposomes to mice via venous infusion. Following infusion, we will measure blood pressure, kidney function, and kidney histopathology to determine if any adverse effects are present. The NRSA award will provide me a unique opportunity to leverage the existing research and development infrastructure of my mentor, Dr. Mark Gladwin, to gain advanced training in animal models of disease, protein engineering, and heme biochemistry. The resources and experience of my mentor Dr Gladwin, an expert in NO and globin biochemistry, as well as the translational development of drugs from the laboratory to the clinic, combined with the expansive array of resources available at the University of Pittsburgh, will allow me to develop new translational research skills with a specific focus on development of novel therapeutic strategies. The NRSA will also support my completion of the requirements for a PhD in Bioengineering and defense of my thesis, as well as my return to medical school, where I will begin to develop my clinical skillset in preparation for an eventual career in academic medicine.
This proposal explores the potential of the vertebrate heme globins cytoglobin and neuroglobin to form the basis of a new generation of protein-based oxygen carriers. Using recombinant mutagenesis, thorough in vitro characterization of protein function, and an animal model of heme globin infusion, this proposal seeks to show that these proteins can reduce the renal toxicity and hypertension associated with HBOCs. Completion of these aims will also reveal additional information about the structure-function relationships of these proteins, potentially informing development of other globin-based therapeutics.
| Amdahl, Matthew B; DeMartino, Anthony W; Tejero, Jesús et al. (2017) Cytoglobin at the Crossroads of Vascular Remodeling. Arterioscler Thromb Vasc Biol 37:1803-1805 |
| Amdahl, Matthew B; Sparacino-Watkins, Courtney E; Corti, Paola et al. (2017) Efficient Reduction of Vertebrate Cytoglobins by the Cytochrome b5/Cytochrome b5 Reductase/NADH System. Biochemistry 56:3993-4004 |
