P166Coordination of gtpase and calcium signalling by Rab46 regulates histamine specific weibel palade body trafficking and protects the vasculature from a pro-thrombotic response
P166Coordination of gtpase and calcium signalling by Rab46 regulates histamine specific weibel palade body trafficking and protects the vasculature from a pro-thrombotic response
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P166 Rab46 协调 gtpase 和钙信号传导调节组胺特异性 weibel palade 体运输并保护脉管系统免受促血栓反应
DOI：
10.1093/cvr/cvy060.126
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发表时间：
2018
影响因子：
10.8
通讯作者：
Mckeown L
中科院分区：
文献类型：
--
作者：
Mckeown L
背景：储存在内皮细胞韦贝尔帕拉德体（WPBs）中的促凝剂和促炎介质的释放是有效应对血管损伤的必要条件。然而，不适当和不及时的wpb胞吐可促进心血管疾病中明显的病理性血栓形成环境。免疫原性刺激如组胺也能引起WPB胞吐，导致免疫反应，而不诱导凝血或伤口愈合。虽然胞内Ca2+的动员是WPB胞吐所必需的，但为了产生这些生理上不同的反应，货物的差异释放的机制尚不清楚。最近，我们在内皮细胞中描述了一种新的Rab GTPase (Rab46)，它位于WPBs上，具有GTPase和Ca2+结合活性。目的：探讨Rab46在组胺和凝血酶诱导的白细胞转运中的作用。方法：我们用组胺（免疫原性）或凝血酶（促血栓）处理HUVECs，并使用超分辨率显微镜对WPBs进行成像。采用elisa和western blotting技术检测p -选择素和血管生成素-2。利用特异性靶向siRNA和核苷酸和Ca2+结合位点的定点诱变来确定Rab46依赖性。结果：组胺（30µm）而不是凝血酶（2U/ml）引起了wpb亚群向核周区域的运输，该区域被确定为微管组织中心。组胺诱导的WPB核周转运依赖于Rab46、微管和GTPase。免疫染色显示含有p -选择素和血管生成素-2的WPBs是相互不同的。我们认为，由于组胺和凝血酶反应都需要p选择素依赖的免疫细胞吸引，也许组胺激活Rab46可以拯救大量的WPBs，使其免于分泌p选择素阴性但含有免疫原性反应不需要的货物（如血管生成素-2）。Rab46在含有p -选择素的WPBs中不存在，但在含有血管生成素-2的WPBs中存在（54%+/-11.04总血管生成素-2）。凝血酶和组胺处理HUVECS可独立刺激p -选择素的胞外分泌（对照siRNA: 71.7%+/-9.7: 71.2%+/-10.7; Rab46 siRNA: 80.1%+/-12.2, 70.3%+/-9.7）。P => 0.05差异无统计学意义。n / n = 5/15)。组胺而非凝血酶诱导血管生成素-2/Rab46阳性WPBs的核周定位，抑制血管生成素-2（促血栓介质）的释放。在持续的组胺刺激下，从细胞内储存释放的Ca2+与Rab46的EF-hand结构域结合，介导WPB从核周簇中分散，准备重新使用。结论：这些观察结果表明Rab46是不同WPB货物分泌的关键调节因子，了解Rab46/WPB信号轴可能为心血管疾病的治疗提供新的靶点。
Background: The release of pro-coagulant and pro-inflammatory mediators stored in endothelial Weibel Palade bodies (WPBs) is necessary for an efficient response to vascular injury. However, inappropriate and untimely exocytosis of WPBs can promote the pathological thrombotic environment evident in cardiovascular disease. Immunogenic stimuli such as histamine also evoke WPB exocytosis resulting in an immune response without inducing coagulation or wound healing. Whilst the mobilisation of intracellular Ca2+ is necessary for WPB exocytosis, the mechanisms underlying differential release of cargo in order to produce these physiologically distinct responses are poorly understood. Recently, we described a novel Rab GTPase (Rab46) in endothelial cells that has GTPase and Ca2+ binding activities and is located on WPBs.Aim: To investigate the role of Rab46 in histamine and thrombin evoked trafficking of WPBs.Methods: We treated HUVECs with either histamine (immunogenic) or thrombin (pro-thrombotic) and imaged WPBs using Super-Resolution microscopy. ELISAs and western blotting techniques were used to measure P-selectin and angiopoietin-2. Rab46 dependence was determined using specifically targeted siRNA and site directed mutagenesis of nucleotide and Ca2+ binding sites.Results: Histamine (30µm) but not thrombin (2U/ml) provoked trafficking of a subset of WPBs to a perinuclear region determined to be the microtubule-organising centre. Histamine induced WPB perinuclear trafficking was Rab46, microtubule and GTPase dependent. Immunostaining revealed that WPBs containing P-selectin and angiopoietin-2 were mutually distinct. We considered that as histamine and thrombin responses both require P-selectin dependent attraction of immune cells, maybe the activation of Rab46 by histamine rescues a population of WPBs from being secreted that are P-selectin negative but contain cargo not required for an immunogenic response (such as angiopoietin-2). In accordance, Rab46 was absent from WPBs containing P-selectin but was localised to a population of WPBs containing angiopoietin-2 (54%+/-11.04 total angiopoietin-2). Treatment of HUVECS with both thrombin and histamine stimulate the exocytosis of P-selectin independently of Rab46 (control siRNA: 71.7%+/-9.7: 71.2%+/-10.7; Rab46 siRNA: 80.1%+/-12.2, 70.3%+/-9.7 respectively. p=> 0.05 no significant difference. n/N= 5/15). Histamine but not thrombin induced a perinuclear localisation of angiopoietin-2/Rab46 positive WPBs, inhibiting the release of angiopoietin-2, a pro-thrombotic mediator. Under continued histamine stimulation Ca2+ released from intracellular stores binds to the EF-hand domain of Rab46 mediating WPB dispersal from the perinuclear clusters ready for re-use.Conclusions: These observations indicate Rab46 as a key regulator of differential WPB cargo secretion and understanding the Rab46/WPB signalling axis could provide novel therapeutic targets for cardiovascular disease.