Andrikopoulos Petros, Kieswich Julius, Harwood Steven M, Baba Akemichi, Matsuda Toshio, Barbeau Olivier, Jones Keith, Eccles Suzanne A, Yaqoob Muhammad M
From Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary's University of London, London EC1M 6BQ, United Kingdom,
From Translational Medicine and Therapeutics, William Harvey Research Institute, Barts and the London School of Medicine, Queen Mary's University of London, London EC1M 6BQ, United Kingdom.
J Biol Chem. 2015 Jul 24;290(30):18412-28. doi: 10.1074/jbc.M114.628156. Epub 2015 May 15.
Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.
凝血酶通过激活蛋白酶激活受体（PARs）作用于内皮细胞。在病理状态下，内皮凝血酶 - PAR系统失调，导致屏障功能丧失以及出现促炎和促血管生成的内皮表型。我们最近报道，以Ca(2+)内流（反向）模式运行的离子转运体Na(+)/Ca(2+)交换器（NCX）可促进血管内皮生长因子刺激的原代人血管内皮细胞中的ERK1/2激活和血管生成。在此，我们研究了通过NCX的Ca(2+)内流是否参与了凝血酶诱导的ERK1/2激活、血管生成和内皮屏障功能障碍。反向模式NCX抑制剂和RNAi介导的NCX1敲低可减弱凝血酶或主要内皮凝血酶受体PAR - 1激动剂诱导的ERK1/2磷酸化。相反，通过抑制Na(+)-K(+)-ATP酶活性来促进反向模式NCX可增强ERK1/2激活。反向模式NCX抑制剂和NCX1 siRNA可抑制凝血酶诱导的原代人血管内皮细胞血管生成，以增殖和管状分化来量化。反向模式NCX抑制剂或NCX1敲低可在体外凝血酶刺激时保持屏障完整性。此外，反向模式NCX抑制剂SEA0400可抑制伊文思蓝白蛋白向肺和肾脏的渗出，并减轻用PAR - 1肽激活剂攻击的小鼠肺部的水肿形成和ERK1/2激活。从机制上讲，凝血酶诱导的ERK1/2激活需要NADPH氧化酶2介导的活性氧（ROS）产生，而反向模式NCX抑制剂和NCX1 siRNA可抑制凝血酶诱导的ROS产生。我们提出，反向模式NCX是一种新机制，通过以ROS依赖的方式介导ERK1/2激活，促进凝血酶诱导的血管生成和高通透性。靶向反向模式NCX可能对涉及凝血酶信号失调的病理状态有益。
