Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK.
School of Science, Engineering & Design, Teesside University, Stephenson Street, Middlesbrough TS1 3BA, UK.
J Cell Sci. 2021 Apr 1;134(7). doi: 10.1242/jcs.253179. Epub 2021 Apr 13.
Many neuronal and retinal disorders are associated with pathological hyperpermeability of the microvasculature. We have used explants of rodent retinae to study acute neurovascular permeability, signal transduction and the role of AMP-activated protein kinase (AMPK). Following stimulation with either vascular endothelial growth factor (VEGF-A) or bradykinin (BK), AMPK was rapidly and strongly phosphorylated and acted as a key mediator of permeability downstream of Ca2+. Accordingly, AMPK agonists potently induced acute retinal vascular leakage. AMPK activation led to phosphorylation of endothelial nitric oxide synthase (eNOS, also known as NOS3), which in turn increased VE-cadherin (CDH5) phosphorylation on Y685. In parallel, AMPK also mediated phosphorylation of p38 MAP kinases (hereafter p38) and HSP27 (HSPB1), indicating that it regulated paracellular junctions and cellular contractility, both previously associated with endothelial permeability. Endothelial AMPK provided a missing link in neurovascular permeability, connecting Ca2+ transients to the activation of eNOS and p38, irrespective of the permeability-inducing factor used. Collectively, we find that, due to its compatibility with small molecule antagonists and agonists, as well as siRNA, the ex vivo retina model constitutes a reliable tool to identify and study regulators and mechanisms of acute neurovascular permeability.
许多神经元和视网膜疾病都与微血管的病理性高通透性有关。我们使用啮齿动物视网膜的外植体来研究急性神经血管通透性、信号转导以及 AMP 激活的蛋白激酶 (AMPK) 的作用。在受到血管内皮生长因子 (VEGF-A) 或缓激肽 (BK) 刺激后，AMPK 迅速且强烈地磷酸化，并作为 Ca2+下游通透性的关键介质起作用。相应地，AMPK 激动剂能强烈诱导急性视网膜血管渗漏。AMPK 的激活导致内皮型一氧化氮合酶 (eNOS，也称为 NOS3) 的磷酸化，进而增加 VE-钙黏蛋白 (CDH5) 在 Y685 上的磷酸化。平行地，AMPK 还介导 p38 MAP 激酶 (以下简称 p38) 和 HSP27 (HSPB1) 的磷酸化，表明它调节细胞旁连接和细胞收缩性，这两者先前都与内皮通透性有关。内皮 AMPK 为神经血管通透性提供了一个缺失的环节，将 Ca2+ 瞬变与 eNOS 和 p38 的激活联系起来，而与使用的通透性诱导因子无关。总的来说，我们发现，由于其与小分子拮抗剂和激动剂以及 siRNA 的兼容性，离体视网膜模型是一种可靠的工具，可以识别和研究急性神经血管通透性的调节剂和机制。
