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

剖析抗神经酰胺 scFv 血管缓解辐射胃肠道综合症的作用

• 批准号: 9981619
• 负责人: Richard N Kolesnick
• 金额: $ 59.29万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9981619
• 关键词: Acute; Affect; Alleles; Apoptosis; Apoptotic; Attenuated; Binding; Biological; Biological Assay; Blood Circulation; Blood Vessels; Cell Survival; Cell membrane; Cells; Ceramides; Cessation of life; Clinical; Columnar Cell; Complement; Couples; Coupling; Data; Dependence; Development; Dose; Endothelial Cells; Endothelium; Epithelial; Epithelial Cells; Epithelium; Event; Evolution; Functional disorder; Gastrointestinal tract structure; Generations; Genetic; Genotype; Growth; Immunoglobulin M; Infection; Injury; Intestines; Ionizing radiation; Kinetics; Knock-out; Knowledge; LGR5 gene; Laboratories; LacZ Genes; Lipids; LoxP-flanked allele; Measures; Mediating; Mitotic; Mucous Membrane; Mus; National Institute of Allergy and Infectious Disease; Natural regeneration; Nuclear; Organoids; Outcome; Phase; Play; Process; Publishing; Radiation; Radiation Toxicity; Reporter; Reporting; Resistance; Role; Second Messenger Systems; Signal Transduction; Small Intestines; Small intestine mucous membrane; Sphingomyelins; Structure of intestinal gland; Surface; Syndrome; Villus; acid sphingomyelinase; base; cell injury; cell regeneration; endothelial dysfunction; homologous recombination; improved; interleukin-22; new technology; potential biomarker; prevent; progenitor; radiation mitigation; radiation mitigator; radiation response; radiation-induced injury; radioresistant; regenerative; response; stem cells; subcutaneous; tool

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.

项目概要/摘要 对于急性放射性胃肠道综合症（RGS），目前尚无有效的对策。这里提出的研究 将为我们持续努力开发抗神经酰胺抗体作为基于机制的方法提供支持 减轻 RGS 致死率。 RGS 是由于隐窝/绒毛单位遭到破坏、粘膜完整性丧失以及 常驻肠道细菌菌群感染。最近的数据表明 RGS 病理生理学涉及 位于 Lieberkühn 隐窝底部的小肠干细胞 (ISC) 池，称为隐窝基底 柱状细胞（CBC）。我们最近表明 CBC 比转运放大祖细胞具有更强的抗辐射能力 或分化的绒毛细胞，通过同源重组有效地保护自身。此外，加拿大广播公司 耗尽是双相后电离辐射 (IR)，因为细胞凋亡发生在生长停滞期间的前 24 小时内， 随后，在 CBC 开始重新生成后的快速再生阶段，在 24-48 小时内发生有丝分裂死亡。 骑自行车。细胞凋亡和有丝分裂死亡导致的 CBC 消耗先于第 2.5 天至隐窝的物理溶解 3.0 后红外。此外，第 2 天的 CBC 存活率可预测第 3.5 天的隐窝再生（通过 Withers 和 Elkind 的克隆形成分析以及 RGS 致死率。除了直接的 ISC 损害外，我们的实验室还提出 IR 引起的小肠微血管损伤在结果中起着重要作用。具体来说，我们 提出 IR 导致酸性鞘磷脂酶 (ASMase) 在几分钟内释放到外内皮血浆 膜，在那里它找到其底物鞘磷脂并产生促凋亡的第二信使 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，并且 微血管损伤与直接 ISC 损伤的耦合协调决定了 ISC 的存活。此外，我们 最近报道我们的抗神经酰胺 2A2 Ab 与受辐射的内皮表面上的神经酰胺结合，防止 形成内皮死亡所需的富含神经酰胺的平台，从而保护小鼠免受 RGS 侵害 杀伤力。在未发表的研究中，我们发现内皮细胞凋亡的第二波延长波发生在高电压后 剂量IR伴随ASMase分泌到小鼠的体循环中，持续4天 表明正在进行的血管损伤/功能障碍。在这里，我们将在 3 个具体目标中详细介绍这种血管 综合征，确定其在 IR 后 CBC 死亡中的作用，并表明单链可变片段 (scFv) 皮下注射抗神经酰胺抗体减弱它，减轻 CBC 消耗并提高小鼠存活率。使用 新的遗传和生物工具，我们将正式证明 scFv 通过抑制神经酰胺来减轻 RGS 在内皮细胞上产生，但不在肿瘤实质细胞上产生。一旦 scFv 缓解机制确定 我们将把它与 IL-22 结合起来，IL-22 是我们最近展示的一种可以改善 IR 后 CBC 再生的药物。 目前缺乏有关小肠辐射后立即环境的信息 粘膜。这里的研究应该填补微血管损伤对不断发展的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 致死率。