Regulation of lung vascular permeability by lysosomal trafficking

溶酶体运输调节肺血管通透性

• 批准号: 326606907
• 负责人: Professor Dr. Wolfgang Kübler
• 金额: --
• 依托单位: Institut für Pharmakologie und Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/326606907?language=en
• 关键词: Regulation; lung; vascular; permeability; lysosomal

The inflammatory mediator platelet-activating factor (PAF) causes lung edema by increasing vascular permeability through parallel activation of acid sphingomyelinase (ASMase) and cyclooxygenase (COX). In previous funding periods we have outlined the signaling pathway by which ASMase and COX trigger an increase in endothelial Ca2+ with subsequent barrier failure and edema formation. As such we could demonstrate that ASMase causes recruitment of eNOS and the Ca2+ channel TRPC6 into caveolae of endothelial cells, where TRPC6 is activated through the COX-dependent formation of prostaglandin E2 and its action on the EP3 receptor, and is in parallel disinhibited through inhibition of eNOS in caveolae. The signaling pathway distal of ASMase has thus been well characterized; yet, the upstream mechanisms by which PAF activates ASMase in endothelial cells remain so far largely unclear. Under basal conditions ASMase is transported, via binding to the mannose-6-phosphate receptor (M6PR), into lysosomes, the acidic milieu of which warrants the optimal pH for ASMase activity. Upon PAF stimulation, ASMase activity increases in caveolae, where ASMase gains direct access to its substrate sphingomyelin in the outer leaflet of the plasma membrane. This scenario, however, requires the maintenance of an acidic milieu in the caveolae, as well as a stable anchoring of the ASMase, which itself does not contain a transmembrane domain, at the outer leaflet of the plasma membrane. Based on our previously published work and new preliminary data we therefore postulate a) that PAF triggers the exocytosis of endothelial lysosomes resulting in the expression of ASMase in caveolae where it is anchored via binding to M6PR, b) that lysosomal exocytosis is mediated via activation of G-proteins and endothelial Ca2+ signaling, and c) that caveolar acidification required for ASMase activation is facilitated by simultaneous exocytosis of lysosomal V1-H+-ATPase and the Cl- channel CFTR.The proposed experiments will be conducted at large in isolated-perfused rat and mouse lungs, with the effects of PAF being analyzed by functional measurements of vascular permeability and edema formation, real-time imaging of endothelial Ca2+ signaling, lysosomal trafficking, and caveolar acidification, as well as proteomic analyses of the caveolar fraction by use of pharmacological interventions and gene-deficient mouse strains. Newly identified pathomechanisms will subsequently be validated in vivo in established models of acute lung injury in the preclinical setting of a mouse intensive care unit.The results of our work are expected to yield novel insights into the regulation and relevance of lysosomal trafficking in the vascular endothelium, and to identify new pharmacological targets for the prevention and treatment of acute lung edema.

炎症介质血小板活化因子(PAF)通过同时激活酸性鞘磷脂酶(ASMase)和环氧合酶(COX)，增加血管通透性，从而导致肺水肿。在之前的资助时期，我们已经概述了ASMase和COX触发内皮细胞钙离子增加、随后的屏障失效和水肿形成的信号通路。因此，我们可以证明ASMase导致eNOS和钙通道TRPC6重新聚集到内皮细胞的小窝中，其中TRPC6通过COX依赖的前列腺素E2的形成和对EP3受体的作用而被激活，并通过抑制小窝中的eNOS而被平行地抑制。因此，ASMase远端的信号通路已经被很好地描述；然而，PAF激活内皮细胞中ASMase的上游机制到目前为止仍然很不清楚。在碱性条件下，ASMase通过与甘露糖-6-磷酸受体(M6PR)结合而运输到溶酶体中，溶酶体的酸性环境为ASMase活性的最适pH提供了保证。在PAF的刺激下，ASMase的活性在小窝中增加，在那里ASMase可以直接接触到质膜外层小叶中的底物鞘磷脂。然而，这种情况需要在小窝中保持酸性环境，以及ASMase的稳定锚定，因为ASMase本身不包含跨膜结构域，在质膜的外层小叶。因此，根据我们以前发表的工作和新的初步数据，我们假设a)PAF通过与M6PR结合而触发内皮溶酶体的胞吐导致ASMase在小窝中的表达，b)溶酶体的胞吐是通过激活G-蛋白和内皮钙信号来介导的，以及c)ASMase激活所需的空泡酸化是通过同时胞吐溶酶体V1-H+-ATPase和氯通道CFTr来促进的。所提出的实验将在隔离灌流的大鼠和小鼠肺中进行，所提出的实验将通过血管通透性和水肿形成的功能测量、内皮细胞钙信号的实时成像来分析PAF的影响溶酶体运输和空泡酸化，以及利用药理学干预和基因缺陷小鼠品系对空泡部分进行蛋白质组学分析。新发现的病理机制随后将在建立的急性肺损伤模型中进行活体验证，该模型位于小鼠重症监护病房的临床前环境中。我们的工作结果有望为血管内皮细胞溶酶体转运的调节和相关性提供新的见解，并为预防和治疗急性肺水肿找到新的药理靶点。