The regulation of pulmonary vascular permeability by PAF : role of caveolae and acid sphingomyelinase

Vascular permeability is a hallmark of pulmonary inflammation. In the lungs increased vascular permeability leads to Acute Lung Injury (ALI) and the Acute Respiratory Distress Syndrome (ARDS), clinical conditions for which mortality remains high. One important mediator that plays a critical role in many experimental models of ALI is the platelet-activating factor (PAF). PAF increases vascular permeability by activation of two lipid modifying enzymes: cyclooxygenase leading to production of PGE2 followed by activation of EP3 receptors and acid sphingomyelinase (ASM) leading to formation of ceramide. The mechanisms by which ASM-metabolites regulate vascular permeability were unknown and were subject of the present studies. The major aim of this work was to investigate the mechanisms by which ASM regulates the PAF-induced increased pulmonary endothelial permeability. Particularly, we focused on the role of NO synthesis and Ca2+ influx in response to PAF. Since caveolae are particularly rich in sphingolipids, we asked whether PAF acts in the realm of caveolae and which role they play in PAF-induced edema formation. Therefore, it was another goal of this thesis to establish a method for the isolation of caveolae from intact endothelial cells of isolated perfused rat lungs and to characterise these caveolae in PAF-treated lungs. In short summary, we found PAF treatment induced edema formation and decreased endothelial NO formation. In caveolae fractions from pulmonary vascular endothelial cells (isolated from perfused rat lungs), the abundance of caveolin-1 and eNOS increased rapidly after PAF perfusion. Restoration of endothelial NO levels with exogenous NO donor mitigated the PAF-induced edema. PAF treatment increased the ASM activity in caveolar fractions and perfusion with ASM decreased endothelial NO production. Pharmacological inhibition of the ASM pathway with imipramine, D609 or dexamethasone blocked the PAF-induced increase of caveolin-1 and eNOS in caveolae, as well as the decrease in NO production and edema formation. PAF treatment also increased endothelial [Ca2+]i. This response was abrogated by Imipramine, the ASM-inhibitor ARC39 and in ASM-deficient mice. Conversely, endothelial [Ca2+]i was increased by direct perfusion with ASM. Both the PAF-induced [Ca2+]i increase and edema formation was blocked by the unselective TRPC inhibitor SKF96365. Screening for a variety of TRPC channels in caveolar fractions from pulmonary vascular endothelial cells revealed that PAF increased the abundance of TRPC6 channels, and this response was abrogated ARC39. The PAF-induced increase in endothelial [Ca2+]i and the edema formation was absent in TRPC6-deficient mice. We conclude that PAF causes ASM-dependent enrichment of caveolin-1 and TRPC6-channels in caveolae of endothelial cells. This leads to decreased NO production and increased Ca2+ levels. We propose that the decreased NO levels sensitize endothelial cells for Ca2+-dependent mechanisms of endothelial permeability