Regulation of vascular permeability by sphingolipids

促炎介质血小板活化因子（PAF）通过增加由酸性鞘磷脂酶（ASM）和环氧合酶介导的血管通透性而引起肺水肿。在当前的资助期内，我们已经表明，ASM介导小窝蛋白-1，eNOS（内皮NO合酶）和TRPC 6-通道向小窝的募集，这导致内皮NO合成减少，同时细胞溶质钙增加。这些结果表明，PAF引起水肿的同时减少NO和增加钙离子水平在内皮细胞的ASM依赖性机制。虽然这些正在进行的研究已经确定了ASM增加肺血管通透性的信号步骤，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。这些研究中的大多数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诱导的小窝改变，我们将通过质谱分析通过鞘脂和蛋白质的梯度密度离心获得的小窝级分。此外，我们将讨论前列腺素E2的相互作用，这是负责PAF诱导的水肿的ASM独立的一部分，与招聘或激活的TRP通道。B）为了理解PAF如何激活ASM，我们将关注src激酶、cAMP依赖性信号传导和ASM从溶酶体的可能易位。此外，我们将解决甘露糖-6-磷酸受体作为细胞外ASM的锚的作用。这些研究将有助于我们了解炎症性疾病的肺血管通透性的调节，并将有助于确定新的药理学治疗目标。

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.