P136	CD40 stimulation inhibits VEGF induced endothelial cell migration.
U.Thanabalasingam, D.Stibenz, S.Goetze, Ph.Stawowy, M.Roser, H.Kallisch, K.Graf, E.Fleck, M.Gräfe
Deutsches Herzzentrum Berlin, Berlin, DE.

Background: CD40 is a cell surface receptor found on B-cells, smooth muscle cells and endothelial cells and belongs to the TNF receptor family. It mediates inflammatory responses. Increased expression is found in atherosclerotic lesions, but the existing data on its impact on angiogenesis, an important mechanism in the progression of atherosclerosis, are controversial. The present study was designed to investigate the effect of CD40 stimulation on migration of endothelial cells. Methods: Isolated human umbilical vein endothelial cells were stimulated either with TNFalpha for 4 hrs or with stably CD40-Ligand (CD154) expressing mouse myeloma cells (P3xTBA7) and wildtype cells (P3xWT) for 4 or 24 hrs. Subsequently, migration of endothelial cells towards the chemotactic stimulus VEGF (10ng/ml) was examined in transwell cell culture chambers with polycarbonate membranes that were coated with gelatine. Since the signaling pathways PI3-Kinase ->Akt->eNOS and ERK1/2 MAP-Kinase are involved in cell migration, activity of ERK1/2, Akt, eNOS and PTEN, a negative regulator of PI3K->Akt singaling were assessed by Western Blotting. Proper stimulation of endothelial cells were examined by measuring expression of cell adhesion molecules E-Selectin, VCAM-1 and ICAM-1 by flow cytometry. Results: Stimulation of endothelial cells with TNFalpha or CD40-Ligand positive cells for 4 hrs had no effect on VEGF induced migration of endothelial cells but induced expression of endothelial cells. Incubation with CD40-Ligand positive cells for 24 hrs completely inhibited VEGF induced migration to levels of unstimulated cells (424 ± 17 cells vs.105 ± 9 cells; VEGF vs CD40Ligand+VEGF; p < 0.01). The specific CD40-Ligand effect was demonstrated by using monoclonal anti-CD40Ligand antibodies (230 ± 14 cells; p < 0.01vs CD40L+VEGF)and mock treatment with wildtype cells(256 ± 13 cells; p < 0.01 vs CD40L+VEGF). VEGF induced phosphorylation of Akt and eNOS were significantly inhibited by stimulation with CD40-Ligand positive cells wheras activity of PTEN and ERK1/2 remained unchanged. Conclusion: CD40 stimulation for a short time has no significant effect on migration of endothelial cells. 24h stimulation inhibits endothelial cell migration significantly and reduces phsphorylation of Akt and eNOS, while PTEN and ERK1/2 remain unchanged.

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