Anti-ceramide Ab Preserves Intestinal Stem Cells and Prevents GI-ARS and GI-DEARE - There are no effective countermeasures for the Gastrointestinal-Acute Radiation Syndrome (GI-ARS). Studies proposed here will support our ongoing effort to develop an anti-ceramide Ab as a mechanism-based approach to mitigate GI-ARS morbidity and mortality. GI-ARS results from destruction of crypt/villus units, loss of mucosal integrity, and infection by resident enterobacterial flora. GI-ARS pathophysiology involves depletion of a small pool of intestinal stem cells (ISCs) residing at the base of the Crypts of Lieberkühn termed crypt base columnar cells (CBCs). We showed CBC depletion post-ionizing radiation (IR) occurs over 24-48 h preceding physical crypt dissolution between Day 2.5-3.0 post IR. Further, CBC survival at Day 2 predicts crypt regeneration at Day 3.5, and GI-ARS lethality. Our lab has pioneered the concept that IR releases acid sphingomyelinase (ASMase) within min to outer endothelial plasma membranes, hydrolyzing sphingomyelin to generate pro-apoptotic second messenger ceramide therein, which couples to direct ISC damage to coordinately determine ISC fate. Further, our anti-ceramide Abs bind ceramide on the irradiated endothelial surface protecting mice against GI-ARS lethality. Recently we showed endothelial apoptosis increases 25-fold above background by 4 h post 15 Gy, the GI-ARS LD90 dose in C57BL/6J mice, remains elevated for 36 h, slowly returning to baseline by 84 h. Anti- ceramide delivery at 24 h post IR induces immediate cessation of endothelial apoptosis facilitating Lgr5+ ISC regeneration, preventing lethality. Full autopsies of mice surviving 90 days are nearly normal in 42 organs, including the GI tract. Based on these findings, we initiated studies that address impact of acute anti-ceramide protection on delayed effects of acute radiation exposure (DEARE) to small intestines. We show mice treated with 7-13 Gy whole body irradiation, sublethal GI-ARS doses, plus bone marrow transplantation (BMT) display acutely reversible weight loss and remain asymptomatic for extended periods. However, over time they develop dose-dependent weight loss and at high doses moribundity. Day 200 autopsies reveal dose-dependent villus- crypt blunting and crypt loss, which at high doses are marked, accompanied by vascular irregularities. Anti- ceramide single chain variable fragment treatment at 24 h post IR prevents endothelial apoptosis, facilitating crypt regeneration, preventing delayed weight loss and normalizing Day 200 crypt-villus architecture. Further, using a green fluorescent protein (GFP)-labeled BMT at 20 h post-IR, we discover within hours large numbers of GFP+ endothelial progenitors enter the damaged area, deposit exclusively at the crypt base juxtaposing the CBC compartment, and die by apoptosis. Anti-ceramide provision at 24 h post IR abrogates regenerative endothelial cell apoptosis. We propose protection of regenerative endothelial cells enhances ISC regeneration at 24-72 h post-IR, mitigating GI morbidity/mortality acutely and chronically, to be assessed in 3 Specific Aims. These studies, if successful, will show that acute protection of the microvasculature results in regeneration of the damaged Lgr5+ ISC compartment leading to acute and long-term preservation of small intestinal integrity.
