P280	Vimentin is upregulated in collateral arteries and an important regulotor of endothelial proliferation and migration.
1N.Obermeyer, 2F.Buck, 1W.Ito
1Cardiology, University Medical Center Hamburg, Hamburg, DE; 2Neurobiology, University Medical Center Hamburg, Hamburg, DE.

In order to identify molecules regulated during collateral growth we isolated collateral arteries 12 h, 24 h and 3 days post femoral artery occlusion in the rat. After sequential protein extraction we performed 2-D gel analysis, western blotting and immunohistochemical staining of excised collateral vessels after femoral artery occlusion in the rat. 12 2-D gels (6 experimental (collateral vessel) and 6 control (control vessel) were run in parallel in each experiment in order to guaranty identical running conditions. Image data were analyzed using PDQuest software (Bio-RAD). Regulated protein spots were picked from the gel, trypsin-digested and analyzed by mass spectrometry (MS/MS). Migration was analyzed via time-lapse videomicroscopy. Proliferation of endothelial cells and collateral arteries was determined via FACS analysis respectively immunohistochemically after BrdU labeling. We identified several proteins specifically regulated in migrating endothelial cells and collateral arteries. Among these proteins were the cytoskeletal protein Vimentin that was highly upregulated in endothelial cells and collateral vessels.In order to study the functional relevance of these proteins for endothelial cell migration, proliferation and collateral growth we performed inhibitory experiments with transfection experiments with Vimentin SI RNA. SI RNA uptake was >95%. SI RNA mediated suppression of Vimentin as controlled by western blotting reduced significantly from 0.1±0.019 µm/min to 0.049±0.009µm/min in videomicroscopic analysis. Similar results were obtained in boyden chamber assays. Suppression of vimentin expression also lead to a significant reduction of endothelial proliferation from 36±7% to 19±3%. Our results demonstrate that the intermediate filament vimentin plays an important role during endothelial migration and proliferation, two processes important for vascular growth and vascular remodeling and indicate that the regulation of this protein indeed plays an important role during collateral proliferation in keeping with the tensegrity model of Donald Ingber. Confirmatory in vivo experiments are in progress.

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