Dissecting anti-ceramide scFv vascular mitigation of the Radiation GI Syndrome

项目总结/摘要 急性放射性胃肠道综合征（RGS）尚无有效的防治措施。此处建议的研究 将为我们正在进行的开发抗神经酰胺抗体的努力提供支持，作为一种基于机制的方法， 减轻RGS的致命性。RGS是由于隐窝/绒毛单位的破坏、粘膜完整性的丧失和 由常驻肠细菌植物群感染。最近的数据表明，RGS的病理生理学涉及消耗的一个 位于Lieberkühn隐窝底部的小肠干细胞（ISCs）池，称为隐窝基底 柱状细胞（CBCs）。我们最近发现CBCs比转运扩增祖细胞更耐辐射 或分化的绒毛细胞，通过同源重组有效地保护自身。此外，CBC 消耗是双相的电离辐射后（IR），因为细胞凋亡发生在生长停滞期间的前24小时， 随后在24- 48小时的快速再生期内发生有丝分裂死亡，一旦CBCs开始再生， 骑自行车在第2.5- 2.6天之间，细胞凋亡和有丝分裂死亡导致的CBC耗竭先于隐窝的物理溶解。 3.0此外，第2天的CBC存活率预测了第3.5天的隐窝再生， Withers和Elkind的克隆形成试验和RGS致死率。除了直接的ISC损伤，我们的实验室提出 IR诱导的小肠微血管损伤在结果中起重要作用。我们特别 建议IR在分钟内导致酸性鞘磷脂酶（ASMase）释放到外内皮血浆 膜，在那里它找到它的底物鞘磷脂，并产生促凋亡第二信使 神经酰胺神经酰胺在内皮表面组装介导细胞凋亡的信号平台， 微血管损伤与直接ISC损伤的结合共同决定了ISC的存活。我们还 最近报道，我们的抗神经酰胺2A 2抗体结合神经酰胺的照射内皮细胞表面，防止 形成内皮细胞死亡所需的富含神经酰胺的平台，从而保护小鼠免受RGS 杀伤力在未发表的研究中，我们发现在高血压后， 持续4天的剂量IR伴随ASMase分泌进入小鼠的体循环 表明正在进行的血管损伤/功能障碍。在这里，在3个具体目标，我们将详细介绍这种血管 综合征，确定其在CBC死亡后IR中的作用，并显示 皮下注射抗神经酰胺抗体减弱它，减轻CBC消耗和提高小鼠存活率。使用 新的遗传学和生物学工具，我们将正式证明scFv通过抑制神经酰胺来减轻RGS 产生于内皮细胞而非肿瘤实质细胞。一旦scFv缓解的机制是 我们将联合收割机与IL-22联合使用，IL-22是我们最近发现的一种改善IR后CBC再生的药物。 目前缺乏关于小肠放射后即刻环境的信息。 粘膜这些研究应该填补关于微血管损伤对粘膜损伤影响的空白 并增加我们的知识，以优化抗神经酰胺策略，以减轻RGS的致命性。

Project Summary/Abstract There are no effective countermeasures for the acute Radiation GI Syndrome (RGS). Studies proposed here will provide support for our ongoing effort to develop an anti-ceramide Ab as a mechanism-based approach to mitigate RGS lethality. The RGS results from destruction of crypt/villus units, loss of mucosal integrity, and infection by resident enterobacterial flora. Recent data indicate RGS pathophysiology involves depletion of a pool of small intestinal stem cells (ISCs) residing at the bottom of the Crypts of Lieberkühn termed crypt base columnar cells (CBCs). We recently showed CBCs to be more radioresistant than transit amplifying progenitors or differentiated villus cells, protecting themselves effectively by homologous recombination. Further, CBC depletion is biphasic post-ionizing radiation (IR) as apoptosis occurs in the first 24h during growth arrest, followed by mitotic death at 24-48h during the rapid regenerative phase that ensues once CBCs begin re- cycling. CBC depletion by apoptosis and mitotic death precede physical dissolution of crypts between Day 2.5- 3.0 post IR. Further, CBC survival at Day 2 predicts both crypt regeneration at Day 3.5 measured by the Clonogenic Assay of Withers and Elkind, and RGS lethality. In addition to direct ISC damage, our lab proposed that IR-induced injury to small intestinal microvasculature plays a prominent role in outcome. Specifically, we propose IR causes release within min of acid sphingomyelinase (ASMase) to the outer endothelial plasma membrane, where it finds its substrate sphingomyelin and generates the pro-apoptotic second messenger ceramide. Ceramide assembles a signaling platform on the endothelial surface that mediates apoptosis, and the coupling of microvessel injury to direct ISC damage coordinately determines ISC survival. Further, we recently reported our anti-ceramide 2A2 Ab binds ceramide on the irradiated endothelial surface, preventing formation of ceramide-rich platforms required for endothelial death, thereby protecting mice against RGS lethality. In unpublished studies, we show a 2nd prolonged wave of endothelial apoptosis occurs post high dose IR accompanied by ASMase secretion into the systemic circulation of mice that persists for 4 days indicative of ongoing vascular damage/dysfunction. Here, in 3 specific aims we will detail this vascular syndrome, determine its role in CBC demise post IR, and show that a single chain variable fragment (scFv) of anti-ceramide Ab injected s.c. attenuates it, mitigating CBC depletion and improving mouse survival. Using new genetic and biologic tools we will formally show that the scFv mitigates the RGS by inhibiting ceramide generated on endothelial but not tumor parenchymal cells. Once the mechanism of scFv mitigation is delineated we will combine it with IL-22, an agent recently shown by us to improve CBC regeneration after IR. There is currently a dearth of information regarding the immediate post-radiation milieu of the small intestinal mucosa. Studies here should fill in gaps regarding impact of microvascular damage on evolving mucosal injury and increase our knowledge to optimize anti-ceramide strategies to mitigate RGS lethality.