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.

项目摘要/摘要 急性辐射GI综合征（RGS）没有有效的对策。这里提出的研究 将为我们持续开发抗神经酰胺AB作为基于机制的方法的努力提供支持 减轻RGS致死性。 RGS是由于地下室/绒毛单位的破坏，粘膜完整性的丧失以及 居民肠杆菌菌群感染。最新数据表明RGS病理生理学涉及耗尽 位于Lieberkühn地下室底部的小肠干细胞（ISC）库称为加密基础 柱状细胞（CBC）。我们最近显示，CBC比过境扩增的祖细胞更具放射性 或分化的绒毛细胞，通过同源重组有效地保护自己。此外，CBC 耗竭是双相电源后辐射（IR），因为在生长停滞期间的前24H中凋亡发生， 然后在快速再生阶段的24-48h处发生有丝分裂死亡，一旦CBC开始重新生成 骑自行车。 CBC通过细胞凋亡和有丝分裂死亡的部署之前，在第2.5天之间的隐窝溶解之前 3.0邮政IR。此外，第2天的CBC存活率在第3.5天都在第3.5天进行了隐窝再生 枯萎和麋鹿的克隆生成测定，以及RGS致死性。除了直接ISC损坏，我们的实验室还提议 IR诱导的小肠造成的肠道损伤在结局中起着重要的作用。具体来说，我们 提案IR导致酸鞘磷脂酶（ASMase）内的最小内皮血浆释放 膜，找到其底物鞘磷脂并生成促凋亡的第二质体 神经酰胺。神经酰胺在介导凋亡的内皮表面上组装一个信号平台，并 微血管损伤以直接ISC损伤的耦合共同确定ISC存活。此外，我们 最近报道了我们的抗神经酰胺2A2 AB在辐照的内皮表面上结合神经酰胺，以防止 内皮死亡所需的神经酰胺丰富平台的形成，从而保护小鼠免受RGS的侵害 致死性。在未发表的研究中，我们显示了第二次延长的内皮细胞凋亡波在很高的情况下发生 通过ASMase分泌到持续4天的小鼠系统循环中完成的剂量IR 表示正在进行的血管损伤/功能障碍。在这里，在3个特定目标中，我们将详细介绍这个血管 综合征，确定其在IR后CBC Demise中的作用，并表明单个链可变片段（SCFV） 抗神经酰胺AB注射的S.C.减轻它，减轻CBC部署并改善小鼠的生存率。使用 新的遗传和生物学工具我们将正式表明SCFV通过抑制神经酰胺来减轻RGS 在内皮而不是肿瘤副群细胞上产生。一旦缓解SCFV的机制 划定的我们将与IL-22结合使用，IL-22是我们最近显示的旨在改善IR后CBC再生的代理。 目前，有关小肠的直接放射后环境的信息死亡 粘膜。这里的研究应填补有关微血管损伤对不断发展的粘膜损伤的影响的空白 并提高我们的知识以优化抗神经酰胺策略以减轻RGS的致命性。