Radiation-induced gastrointestinal syndrome (RIGS) results from a combination of direct cytocidal effects on intestinal crypt and endothelial cells and subsequent loss of the mucosal barrier, resulting in microbial infection, septic shock and systemic inflammatory response syndrome (SIRS). The mammalian intestinal mucosa is a rapidly proliferating tissue and is a model for tissue parenchymal cells originating via hierarchical cell proliferation and differentiation from Intestinal stem cells (ISC), located in the crypt. The intestine is also a model for an """"""""early responding"""""""" tissue that is frequently targeted by DNA damaging agents, such as, ionizing radiation (IR), resulting in acute toxicity of radiation-induced gastrointestinal syndrome (RIGS). Since IR depletes ISC clonogens in a dose-dependent stochastic fashion, we hypothesized that RIGS is induced by ISC depletion and signaling defects of IR-damaged ISC niche, thus providing an ideal model system to study ISC growth and differentiation in response to radiation-induced intestinal injury. IR induces apoptosis of ISCs, crypt endothelial cells and enterocytes within hours. We rationalized that the acute loss of cells in situ requires rapid compensation of their functions and this was best achieved with cell replacement therapies, e.g., blood transfusion for hemorrhage or bone marrow transplantation for cytoablative damage. As such, acute gastrointestinal (GI) irradiation exposure will induce a dose-dependent cell death in the normally rapidly proliferating intestinal enterocyte and ISC and endothelial cell compartments. The stroma of solid organs contains a variety of supporting cells, such as mesenchymal-derived cells, microvascular endothelial cells, macrophages and lymphocytes. These stromal cells provide the niche and could supply critical growth factor/signals for ISC regeneration. For example, upon intestinal mucosal disruption, resident macrophages in the intestinal submucosal layers are activated by pathogen-derived ligands for Toll-like receptors (TLR) and upon activation, macrophages act as """"""""mobile cellular transceivers"""""""" that transmit regenerative signals to ISCs. We thereby propose a combination of ISC regenerative therapy with systemic administration of growth factors and cell replacement strategy to salvage GI function post-radiation exposure. In order to develop an ISC-based therapeutic strategy for RIGS, we hypothesized that various combinations of: a) intestinal stem cell growth factor, R-spondin1 (R-spo1), b) activation of Toll-like receptor signaling in resident macrophages and ISC, and c) transplantation of bone marrow-derived endothelial and mesenchymal stem cells to restore the IR-damaged ISC niche would augment host ISC regeneration and repair of the ISC niche in irradiated mice, thus providing protection and mitigation from RIGS. In order to test our hypotheses, we propose studies with the following specific aims:
Specific Aim I. To investigate whether- a) an intestinal stem cell growth factor, R-spondin 1 (expressed by a recombinant adenovirus and/or recombinant R-SPO1 protein obtained from Nuvelo Corporation, San Carlos, CA), and/or, b) stimulation of TLR signaling pathway can augment intestinal crypt cell regeneration and support a cell transplant strategy to ameliorate RIGS.
Specific Aim II. To examine whether transplantation of bone marrow-derived adherent mesenchymal stromal and endothelial progenitor cells can induce regeneration of the intestinal mucosa and further augment the protective effects in the presence of these growth factors. We will examine whether each of the above treatments can enhance initial engraftment, proliferation and the level of repopulation of cellular transplantation strategies, alone or in combination, to overcome RIGS and host lethality.
Radiation-induced gastrointestinal syndrome (RIGS) results from a combination of direct cytocidal effects on intestinal crypt and endothelial cells and subsequent loss of the mucosal barrier, resulting in microbial infection, septic shock and systemic inflammatory response syndrome. Irradiation induces apoptosis of crypt endothelial cells, intestinal stem cells (ISC) and enterocytes within hours. We rationalized that the acute loss of cells in situ requires rapid compensation of their functions and this was best achieved with cell replacement therapies, e.g., blood transfusion for hemorrhage. The stroma of solid organs contains a variety of supporting cells, such as, mesenchymal and microvascular endothelial cells, macrophages and lymphocytes. These stromal cells provide the niche and could supply critical growth factor/signals for ISC regeneration. For example, upon intestinal mucosal disruption, resident macrophages in the intestinal submocosal layers are activated by pathogen-derived ligands for Toll-like receptors (TLR) and transmit regenerative signals to ISCs. We thereby propose intestinal regenerative therapy with a combination of systemic administration of growth factors and cell replacement therapy to salvage GI function post-radiation exposure. In order to develop an stem cell-based therapeutic strategy for RIGS, we hypothesized that combinations of: a) intestinal stem cell growth factor, R-spondin1 (R-spo1), b) TLR ligands, and c) transplantation of bone marrow-derived endothelial progenitor cells (EPC) and mesenchymal stem cells (MSC) would restore the IR-damaged ISC niche, protect against IR-induced cell death and provide growth signals for host ISC regeneration, thus providing protection and mitigation from RIGS. Experiments will be designed with the following specific aims: Aim I. To investigate whether- a) R-spondin 1, and/or, b) stimulation of TLR signaling can augment intestinal crypt cell regeneration and support ISC regeneration to ameliorate RIGS. Aim II. To examine whether transplantation of bone marrow-derived MSCs and EPCs can induce regeneration of the intestinal mucosa and further augment the protective effects in the presence of these growth factors. Relevance: There are currently no approved therapeutic treatments to effectively protect first responders from Acute Radiation Syndrome (ARS) or to alleviate ARS in victims of radiological disaster. To date, only Ethyol (Amifostine) has been approved by the FDA for clinical radioprotection of salivary glands, when used prior to exposure to external beam radiation therapy for H&N cancer. Most post-event strategies associated with ARS have been severely limited to within only several hours of the event (with the exception of bone marrow transplantation) and have demonstrated only marginal protection. As such, there is no known post-exposure strategy to rescue/ salvage critical biological elements of RIGS within days after the radiation event has occurred. Our preliminary results demonstrate that intravenous transplantation of bone marrow-derived EPC and MSC, 24 hours after exposure of up to 18 Gy whole abdominal irradiation, can completely protect C57Bl/6 mice from RIGS. Ideal therapy for RIGS would be multimodal that stimulates ISC regeneration and restores the GI mucosal integrity;provides supportive therapy to prevent infection and dehydration;and induces repopulation of the intestinal and mesenchymal stem cells.
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