Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger
Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger
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DOI：
10.1074/jbc.m114.628156
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发表时间：
2015-07-24
影响因子：
4.8
通讯作者：
Yaqoob, Muhammad M.
中科院分区：
文献类型：
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作者：
Andrikopoulos, Petros;Kieswich, Julius;Yaqoob, Muhammad M.
凝血酶通过激活蛋白酶激活受体(PARs)作用于血管内皮细胞。在病理条件下，内皮细胞凝血酶-PAR系统失去调节，导致屏障功能丧失，并形成促炎和促血管生成的内皮细胞表型。我们最近报道，在血管内皮生长因子刺激的原代人血管内皮细胞中，以钙离子内流(反向)模式工作的离子转运体Na+/钙离子交换器(NCX)促进ERK1/2的激活和血管生成。在这里，我们研究了通过NCX的钙离子内流是否参与了ERK1/2的激活、血管生成和凝血酶反应的内皮屏障功能障碍。反向模式的NCX抑制剂和RNAi介导的NCX1基因敲除可减弱凝血酶或主要的内皮凝血酶受体PAR-1激动剂对ERK1/2的磷酸化反应。相反，通过抑制Na+-K+-ATPase活性促进反向模式NCX可增强ERK1/2的激活。反向模式NCX抑制剂和NCX1 siRNA抑制凝血酶诱导的人血管内皮细胞原代血管生成，量化为增殖和小管分化。反向模式的NCX抑制剂或NCX1基因敲除在体外凝血酶刺激下保护屏障的完整性。此外，反向模式的NCX抑制剂SEA0400抑制了埃文斯蓝色白蛋白向肺和肾脏的渗出，并减轻了PAR-1多肽激活剂攻击的小鼠肺中水肿的形成和ERK1/2的激活。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ROS产生，而反向模式的NCX抑制剂和NCX1 siRNA抑制凝血酶诱导的ROS产生。我们认为，反向模式NCX是一种新的机制，通过以ROS依赖的方式介导ERK1/2的激活，从而促进凝血酶诱导的血管生成和高通透性。靶向反向模式的NCX在涉及未受调控的凝血酶信号的病理条件下可能是有益的。
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+/Ca2+ exchanger (NCX) operating in the Ca2+-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca2+ 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.