Neuromuscular Immunopathology Research Laboratory, Department of Neurology, Baylor College of Medicine, Houston, TX 77030-3411, USA.
Microvasc Res. 2012 May;83(3):298-310. doi: 10.1016/j.mvr.2012.01.005. Epub 2012 Feb 2.
Endoneurial microvessels and the perineurium are responsible for maintaining homeostasis in peripheral nerves. Endoneurial endothelial cells form the blood-nerve barrier (BNB). The molecular pathways responsible for endoneurial microvascular barrier formation in humans are not fully understood. We tested the effect of different mitogens on the transendothelial electrical resistance (TEER) of confluent primary human endoneurial endothelial cell (pHEndEC) cultures following serum withdrawal (mimicking diffuse endothelial injury) in vitro. We show that glial-derived neurotrophic factor (GDNF, 1 ng/mL) sufficiently induced a maximal 114.2% recovery in TEER over basal conditions 48 h after serum withdrawal. Solute permeability to high molecular weight dextran was reduced by 52.4% following GDNF treatment. GDNF-mediated increase in TEER was dependent on RET tyrosine-kinase signaling pathways and mildly enhanced by cyclic adenosine monophosphate in combination with maximal concentrations of multiple redundant mitogens. There was no significant increase in adherens or tight junction proteins β-catenin, VE-Cadherin, zona occludens-1 and occludin following GDNF treatment. GDNF induced a small increase in total claudin-5 protein expression without significant increase in messenger RNA or modulation in tyrosine phosphorylation following serum withdrawal. Indirect immunocytochemistry revealed membrane relocation of longitudinal F-actin cytoskeletal filaments in pHEndECs following GDNF treatment, resulting in more continuous intercellular contacts that formed adherens and tight junctions. Together, these results demonstrate a sufficient role for GDNF in human BNB recovery following serum withdrawal in vitro, facilitated primarily by endothelial cell cytoskeletal reorganization. These observations provide insights into the regulation of human BNB function during recovery from peripheral nerve injury.
神经内膜微血管和神经外膜负责维持周围神经的内环境稳定。神经内膜内皮细胞形成血神经屏障（BNB）。人类神经内膜微血管屏障形成的分子途径尚不完全清楚。我们在体外测试了不同有丝分裂原对血清饥饿（模拟弥漫性内皮损伤）后，人原代神经内膜内皮细胞（pHEndEC）培养物中跨内皮电阻（TEER）的影响。我们发现胶质细胞衍生的神经营养因子（GDNF，1ng/mL）在血清饥饿后 48 小时足以诱导 TEER 相对于基础条件的最大 114.2%的恢复。GDNF 处理后，高分子量葡聚糖的溶质渗透性降低了 52.4%。GDNF 介导的 TEER 增加依赖于 RET 酪氨酸激酶信号通路，并且在与最大浓度的多种冗余有丝分裂原组合时轻度增强。GDNF 处理后，β-连环蛋白、VE-钙粘蛋白、闭合蛋白-1 和闭合蛋白的黏附连接和紧密连接蛋白没有明显增加。GDNF 诱导总 Claudin-5 蛋白表达略有增加，但在血清饥饿后，信使 RNA 或酪氨酸磷酸化没有明显增加。间接免疫细胞化学显示，GDNF 处理后 pHEndEC 中的纵向 F-肌动蛋白细胞骨架丝发生膜重定位，导致细胞间接触更加连续，形成黏附连接和紧密连接。总之，这些结果表明，GDNF 在体外血清饥饿后人类 BNB 恢复中起着足够的作用，主要通过内皮细胞细胞骨架重组来实现。这些观察结果为周围神经损伤后人类 BNB 功能的恢复提供了调节机制的见解。
