Import of fluorescent oxidized phospholipids into vascular smooth muscle cells.

Albin Hermetter1, Alexandra Moumtzi1, Michael Trenker1, Sebastian Rhode2, Reinhard Grurl2, Gerhard J. Schütz2

1Department of Biochemistry, Graz University of Technology, Petersgasse 12/2, A-8010 Graz, 2Biophysics Institute, Johannes-Kepler-University Linz, Altenbergerstr.69, A-4040 Linz

Oxidatively fragmented phospholipids including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), present in atherosclerotic lesions, elicit a wide range of pathophysiological responses in the cells of the vascular wall. Fluorescently labeled analogs of these compounds were synthesized to study their uptake, intracellular stability and distribution in vascular smooth muscle cells, (VSMCs). The labeled phospholipids rapidly rapidly moved into VSMC from aqueous phospholipid dispersions or low-density lipoproteins (LDL). According to colocalization experiments with organelle-specific dyes, PGPC derivatives ended up in endosomes and lysosomes. In contrast, POVPC analogs initially accumulated in the plasma membrane as a consequence of covalent reactions with free amino groups of proteins and phospholipids. They reached the cell interior only after prolongued incubation. Time-resolved fluorescence microscopy studies on the single molecule level showed that labelled PGPC rapidly accumulated in small domains of the plasma membrane followed by immediate internalization via endocytosis. In contrast to phospholipids containing two long hydrophobic chains, PGPC is cone-shaped due to the the short polar acyl residue in position sn-2 of glycerol. Colocalisation experiments with fluorescent PGPC and Transferrin/Caveolin as markers for highly curved membranes showed first indications for a higher affinity to such structures. Movement of individual molecules in the plasma membrane confirmed the findings obtained at the ensemble level. Collectively, our data provide evidence that oxidized phospholipids are highly exchangeable between phospholipid surfaces and act on different cellular sites in VSMC within short time, generating multiple platforms from which intracellular signalling is triggered.

Email: albin.hermetter@tugraz.at

Address: Institut fuer Biochemie, Technische Universität Graz, Petersgasse 12/2, A-8010 Graz, Austria