Vascular biology of the human blood-nerve barrier

人类血神经屏障的血管生物学

• 批准号: 8811628
• 负责人: Eroboghene Ekamereno Ubogu
• 金额: $ 33.04万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8811628
• 关键词: Vascular; biology; human; blood; nerve

DESCRIPTION (provided by applicant): Our long-term objective is to study the vascular biology of the human blood-nerve barrier. Specifically, we are interested in studying the role of GDNF and its signaling pathways in restoring blood-nerve barrier function following injury. This project is designed to address a fundamental question: Is GDNF sufficient and essential to the recovery of the blood-nerve barrier following peripheral nerve injury and degeneration? Based on our exciting new preliminary data, we propose the following hypothesis: GDNF sufficiently restores human endoneurial endothelial cell tight junction function following injury primarily by cytoskeletal reorganization that results in more continuous intercellular contacts and fewer intercellular gaps. This occurs without significantly enhancing adherens and tight junction associated protein expression in vitro. GDNF sufficiently restores human endoneurial endothelial cell tight junction barrier function following injury via RET-Tyrosine kinase/ Ras-MAP kinase signaling pathways in vitro. Using a novel conditional GDNF knockout mouse model, we hypothesize that GDNF is essential for the early and persistent restoration of blood-nerve barrier function following sciatic nerve crush injury. GDNF sufficiently restored human blood-nerve barrier resistance following serum withdrawal in vitro in a dose-dependent manner. Our preliminary data specifically implicates the RET-Tyrosine kinase/ Ras-MAP kinase signaling pathways in vitro. Membrane localization of F-actin microfilaments and more continuous intercellular adherens and tight junctions occurred following GDNF treatment of the serum-deprived blood-nerve barrier. We did not observe significant changes in ¿-catenin, VE cadherin, ZO-1, occludin and claudin-5 expression or alterations in claudin-5 tyrosine phosphorylation compared to the untreated injured blood-nerve barrier in vitro. In order to address these hypotheses, we will confirm the sufficient effect of GDNF in restoring blood-nerve barrier function using well-established endothelial barrier experimental assays. Similarly, we will determine that the RET-Tyrosine kinase/ Ras-MAP kinase signaling pathway is responsible for the GDNF-mediated effect following serum withdrawal in vitro, using specific cell-permeable inhibitors in the above experimental assays. Finally, we will evaluate effect of GDNF in restoring blood-nerve barrier function following peripheral nerve injury in vivo using a novel tamoxifen-inducible conditional GDNF knockout mouse strain. Very little is known about the vascular biology of the human blood-nerve barrier. Phenotypic and functional differences between vascular endothelial cells provide the rationale to specifically study the biology of the human blood-nerve barrier in health and disease. Alternations in blood-nerve barrier function have been implicated in many peripheral neuropathies and neuropathic pain. Restoration of blood-nerve barrier function by GDNF could accelerate recovery from these prevalent, disabling medical problems.

描述（由申请人提供）：我们的长期目标是研究人类血神经屏障的血管生物学。具体来说，我们感兴趣的是研究GDNF及其信号通路在损伤后恢复血神经屏障功能中的作用。本项目旨在解决一个基本问题：GDNF是否足以恢复周围神经损伤和变性后的血神经屏障？基于我们令人兴奋的新的初步数据，我们提出了以下假设：GDNF充分恢复人神经内膜内皮细胞紧密连接功能损伤后，主要是通过细胞骨架重组，导致更连续的细胞间接触和更少的细胞间间隙。这在体外没有显著增强粘附蛋白和紧密连接相关蛋白表达的情况下发生。GDNF通过RET-酪氨酸激酶/ Ras-MAP激酶信号通路在体外充分恢复损伤后的人神经内膜内皮细胞紧密连接屏障功能。使用一种新的条件性GDNF基因敲除小鼠模型，我们假设GDNF是必不可少的早期和持续恢复血神经屏障功能后坐骨神经挤压伤。GDNF在体外以剂量依赖性方式充分恢复血清撤回后的人血-神经屏障阻力。我们的初步数据明确暗示了体外RET-酪氨酸激酶/ Ras-MAP激酶信号通路。GDNF处理后，F-肌动蛋白微丝和更连续的细胞间粘附和紧密连接的膜定位发生血清剥夺血神经屏障。我们没有观察到显着的变化，连环蛋白，VE钙粘蛋白，ZO-1，occludin和claudin-5的表达或改变claudin-5酪氨酸磷酸化相比，未经处理的损伤的血神经屏障在体外。为了解决这些假设，我们将使用完善的内皮屏障实验测定来证实GDNF在恢复血-神经屏障功能中的充分作用。同样，我们将在上述实验测定中使用特定的细胞渗透性抑制剂，确定RET-酪氨酸激酶/ Ras-MAP激酶信号传导途径是体外取血清后GDNF介导的作用的原因。最后，我们将使用一种新的他莫昔芬诱导的条件性GDNF基因敲除小鼠品系，评估GDNF在恢复体内周围神经损伤后血神经屏障功能中的作用。对人类血神经屏障的血管生物学知之甚少。血管内皮细胞之间的表型和功能差异提供了专门研究健康和疾病中人类血神经屏障生物学的基本原理。血-神经屏障功能的改变与许多周围神经病和神经性疼痛有关。通过GDNF恢复血神经屏障功能可以加速这些普遍的致残性医学问题的恢复。