The potential severity of a terrorist-driven nuclear or radiological catastrophe may be judged by the recent estimate that 50% of individuals exposed to 400 cGy will die within 60 days unless there is medical intervention. While a percentage of individuals closest to the epicenter of a radiation blast would be killed by incineration or trauma, a significant percentage of victims will be exposed to primarily ionizing radiation exposure. The majority of deaths in these individuals will likely occur due to the deleterious effects of radiation on the bone marrow and immune system. Unfortunately, individuals exposed to radiation doses ranging from 400 cGy - 1000 cGy will frequently die from the sequelae of bone marrow failure (infections, bleeding complications) despite maximal supportive care. Our laboratory has developed methods to cultivate and expand normal murine, primate, and human hematopoietic stem cells via co-culture with primary endothelial cells. Primary ECs support a 1-2 log expansion of human stem cells in the absence of cell-to-cell contact, indicating that EC-derived soluble factors account for this effect. Conditioned medium from primary ECs also supports the functional recovery of BM stem cells following harvest from lethally irradiated animals. In this proposal we aim to characterize the capacity for administered serum free EC-CM alone to rescue in vivo hematopoietic activity and improve survival in animals after exposure to high dose ionizing radiation. Second, we will apply subtractive gene expression analysis and RNA interference methods to identify the EC genes responsible for the uniquely soluble hematopoietic and radioprotective effect produced by ECs. Third, we will apply protein fractionation methods to purify and identify the candidate soluble factors as a complementary and synergistic strategy with our gene expression and siRNA approach. We anticipate that the completion of this project will lead to a deliverable therapeutic not only for radiation sickness, but more broadly, to accelerate hematopoietic recovery following medically indicated therapies (e.g. chemotherapy, radiotherapy, transplantation).
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