Mechanisms driving endothelial angiopoietin-2 expression and vascular dysfunction during pediatric sepsis

这份K08提案描述了一项为期5年的研究培训计划，该计划将为候选人的职业生涯做好准备 一位由美国国立卫生研究院资助的独立翻译科学家专注于脓毒症介导的血管形成的驱动机制 疾病。AIMS的科学前提是血管内皮功能障碍是器官损伤的关键驱动因素 在儿科败血症中。儿童脓毒症的发病率和死亡率仍然高得令人无法接受，尽管在 现代医学，主要是由于在脓毒症期间导致血管疾病的机制方面的知识差距。 血管内皮细胞衍生的血管生成素-2(Ang-2)已成为血管损伤和器官损伤的重要促进剂 脓毒症通过对内皮细胞Tie2受体的拮抗而受损。血浆Ang-2水平为 在脓毒症儿童中显著升高，并与器官损伤和预后的衡量标准有关。 初步数据表明，硫酸乙酰肝素从流动条件下的表面糖唇裂解而来 人肺微血管内皮细胞促进Ang-2的表达，暗示了一种新的范式，通过 ANG-2在脓毒症中表达上调。初步数据和之前的工作也表明，肝脏激酶B1的失活 (LKB1)和下游的腺苷一磷酸活化蛋白激酶(AMPK)可能在这一过程中是不可或缺的 进程。此外，候选人发现Ang-2结合在从血浆中分离的外切体的表面 提示胞外体Ang-2可能在血管疾病和器官病变中起重要作用 脓毒症时的损伤。总之，这些数据支持酶促肝素的新机制假说。 小儿脓毒症时血管内皮细胞的硫酸盐侵蚀可上调Ang-2的表达， 当与外体结合时，具有强烈的血管不稳定作用。为了验证这一假设，在体外、体外和 临床研究将在以下两个目标下进行。目的1：检验糖基化肝素的假说 硫酸盐侵蚀增加血流条件下人肺微血管内皮细胞Ang-2的表达 通过抑制LKB1活性和下游AMPK通路信号传导。目标2：检验假设 胞外体Ang-2(A)是小儿脓毒症器官损伤和临床转归的生物标志物，(B)促进 内皮通透性通过Tie2受体拮抗。利用候选人的综合专业知识 指导团队并利用候选人所在机构的丰富资源，候选人将 培训与实现这些目标密切相关的高级翻译科学技术，包括 微血管流动建模、纳米颗粒跟踪分析和流式细胞仪表征 电池-衬底阻抗传感。拟议的研究计划定义了基准，将促进 候选人的职业发展，并将在提交R01以继续揭示 导致儿童败血症血管疾病的机制，将促进新疗法的发现。

This K08 proposal describes a 5-year research training program that will prepare the candidate for a career as an independent, NIH-funded translational scientist focused on mechanisms driving sepsis-mediated vascular disease. The scientific premise for the aims is that vascular endothelial dysfunction is a key driver of organ injury in pediatric sepsis. Morbidity and mortality remain unacceptably high in pediatric sepsis despite advances in modern medicine, principally due to knowledge gaps in the mechanisms driving vascular disease during sepsis. Endothelial cell-derived angiopoietin-2 (Ang-2) has emerged as a critical promoter of vascular injury and organ impairment in sepsis through its antagonism of the endothelial Tie2 receptor. Plasma Ang-2 levels are significantly elevated in children with sepsis and are associated with measures of organ injury and outcomes. Preliminary data suggest that heparan sulfate cleavage from the surface glycocalyx of flow conditioned primary human lung microvascular endothelial cells promotes Ang-2 expression, implicating a novel paradigm by which Ang-2 is upregulated in sepsis. Preliminary data and prior work also suggest that inactivation of liver kinase B1 (LKB1) and downstream adenosine-monophosphate-activated protein kinase (AMPK) may be integral in this process. Further, the candidate discovered that Ang-2 is bound to the surface of exosomes isolated from plasma of septic children, suggesting that exosomal Ang-2 may significantly contribute to vascular disease and organ injury during sepsis. Together, these data support the novel mechanistic hypothesis that enzymatic heparan sulfate erosion from the endothelial glycocalyx during pediatric sepsis upregulates expression of Ang-2 that, when bound to exosomes, has potent vascular destabilizing effects. To test this hypothesis, in vitro, ex vivo, and clinical studies will be performed in the following two aims. Aim 1: Test the hypothesis that glycocalyx heparan sulfate erosion increases Ang-2 expression from flow conditioned human lung microvascular endothelial cells via attenuated LKB1 activity and downstream AMPK pathway signaling. Aim 2: Test the hypothesis that exosomal Ang-2 (a) is biomarker for organ injury and clinical outcomes in pediatric sepsis and (b) promotes endothelial permeability via Tie2 receptor antagonism. Harnessing the combined expertise of the candidate’s mentoring team and utilizing the wealth of resources available at the candidate’s institution, the candidate will train in advanced translational science techniques germane to the completion of these aims, including microvascular flow modeling, nanoparticle tracking analysis and characterization with flow cytometry, and electric cell-substrate impedance sensing. The proposed research program has defined benchmarks that will facilitate the candidate’s career advancement and will culminate in the submission of an R01 to continue uncovering the mechanisms driving vascular disease in pediatric sepsis that will foster the discovery of novel therapeutics.