Development of angiogenin as a novel radiation medical countermeasure for acute radiation syndrome

PROJECT ABSTRACT Hematopoietic acute radiation syndrome (H-ARS) is characterized by life-threating neutropenia and thrombocytopenia. The functional loss of hematopoietic stem and progenitor cells (HSPCs) are the ultimate cause of the morbidity and mortality. HSPCs of irradiated mice display significantly increased cell cycle, probably as a physiological attempt to maintain HSPC numbers, but undergo fast exhaustion, resulting in a significant decrease in blood cells and bone marrow cellularity. Moreover, HSPC from irradiated mice are severely deficient in engraftment and fail to repopulate bone marrow and peripheral blood cells when transplanted into recipient mice. We have recently found that angiogenin (ANG), a 14 kDa ribonuclease, promotes hematopoietic regeneration by maintaining HSPC quiescence (stemness) and simultaneously stimulating proliferation of myeloid-committed progenitors (MyePros), a unique property that can be harnessed to counteract radiation- induced bone marrow damages in H-ARS. Our preliminary study suggests that ANG mitigates radiation injuries in C57BL/6 mice when administered 24 h post-radiation exposure. We hypothesize that ANG mitigates H-ARS by stimulating MyePro to proliferate to provide a fast replenishment of granulocytes to fight for infections and bleeding, and simultaneously preserving the stemness of HSPCs to prevent stem cell exhaustion thereby maintaining long-term hematopoiesis. The objective of this project is to demonstrate the effectiveness of ANG as a candidate radiation medical countermeasure (MCM) and to elucidate its mechanism of action in promoting hematopoietic regeneration upon radiation injuries. Specific Aim 1 is to obtain pharmacokinetics (PK) of subcutaneously (sc) administered ANG protein in C56BL/6 mice and standard PK parameters will be determined. Specific Aim 2 is to demonstrate radiomitigative potential of ANG in C57BL/6 murine model against total- and partial-body irradiation (TBI and PBI). Three different radiation sources; 60Co gamma radiation for TBI, LINAC for PBI, and reactor for mixed-field (neutron/gamma) will be used to investigate the radiomitigative efficacy. Both female and male mice at the age of 2 – 4, 6 – 8, and 20 – 24 weeks will be used to represent pediatric, adult, and geriatric human population. We will determine the toxicity, safety, optimal dose, and optimal drug administration schedule in relation to irradiation. We will also examine hematopoietic recovery and identify biomarkers for ANG using various omic platforms. Specific Aim 3 is to elucidate the mechanism of action of ANG in mitigating H-ARS. We will characterize the function of ANG in mitigating irradiation-induced damages to HSPC, MyePro, and their subsets. We will determine their cellularity in the bone marrow, apoptosis, ability to form progenitor colonies in methylcellulose, and capacity of reconstituting bone marrow and blood cells in transplantation experiments. We will also examine the effect of ANG on stem and progenitor properties of human CD34+ cord blood cells upon radiation exposure including apoptosis, expression of stemness-related genes, as well as their ability in reconstitution of blood cells in both primary and secondary transplantation in NSG mice.