Regulation of lung vascular permeability by lysosomal trafficking

• Applicants: Professor Dr. Wolfgang Kübler; Professor Dr. Stefan Uhlig
• Subject Area: Anatomy and Physiology ; Anaesthesiology
• Term: from 2017 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 326606907

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.

• DFG Programme: Research Grants