Regulation of vascular permeability by sphingolipids

促炎介质血小板活化因子（PAF）通过酸性鞘磷脂酶（ASM）和环氧合酶介导的血管通透性增加引起肺水肿。在目前的资助期内，我们已经表明，ASM介导小泡蛋白-1、内皮NO合成酶（eNOS）和TRPC6-通道向小泡募集，导致内皮NO合成减少，同时胞质钙增加。这些结果表明，PAF通过asm依赖机制同时降低内皮细胞NO和增加钙水平，从而引起水肿。虽然这些正在进行的研究已经确定了ASM增加肺部血管通透性的信号传导步骤，但我们现在的目标是A)更详细地分析这些机制，B)首先检查导致ASM响应PAF激活的步骤。这些研究大部分将在完整的分离和灌注的大鼠或小鼠肺中进行。A)为了进一步表征paf诱导的小泡改变，我们将用质谱分析梯度密度离心获得的小泡部分鞘脂和蛋白质。此外，我们将讨论前列腺素E2与trp通道的募集或激活之间的相互作用，前列腺素E2负责paf诱导的水肿中与asm无关的部分。B)为了了解PAF如何激活ASM，我们将重点关注src激酶、camp依赖的信号传导以及ASM从溶酶体可能的易位。此外，我们将解决甘露糖-6-磷酸受体作为细胞外ASM锚定的作用。这些研究将有助于我们理解肺炎症性疾病中血管通透性的调节，并有助于确定新的药物治疗靶点。

The pro-inflammatory mediator platelet-activating factor (PAF) causes pulmonary edema by increasing vascular permeability mediated by acid sphingomyelinase (ASM) and cyclooxygenase. In the current funding period we have shown that the ASM mediates recruitment of caveolin-1, eNOS (endothelial NO synthase) and TRPC6- channels to caveolae which leads to a reduction in endothelial NO synthesis in combination with an increase in cytosolic calcium. These findings indicate that PAF causes edema by simultaneously decreasing NO and increasing calcium levels in the endothelium by ASM-dependent mechanisms. While these ongoing studies have identified the signalling steps by which ASM increases vascular permeability in the lungs, we now aim at A) analyzing these mechanisms in greater detail and B) examining the steps that lead to activation of ASM in response to PAF in the first place. Most of these studies will be performed in intact isolated and perfused rat or mouse lungs. A) To further characterize the PAF-induced alterations in caveolae, we will analyze caveolar fractions obtained by gradient density centrifugation for sphingolipids and proteins by mass spectroscopy. In addition, we will address the interaction of prostaglandin E2, which is responsible for the ASM-independent part of PAF-induced edema, with the recruitment or activation of TRP-channels. B) To understand how PAF activates the ASM, we will focus on src kinase, cAMP-dependent signalling and the possible translocation of the ASM from lysosomes. In addition, we will address the role of the mannose-6-phosphate receptor as an anchor for extracellular ASM. These studies will contribute to our understanding of the regulation of vascular permeability in inflammatory disorders of the lung and will help to identify novel pharmacological targets for their treatment.