gms | German Medical Science

Introduction: Human guanylate binding protein-1 (GBP-1) is a large secreted [Ref. 1] GTPase, which exerts potent anti-angiogenic activities in endothelial cells (EC) [Ref. 2], [Ref. 3]. To date, the molecular mechanisms of these anti-angiogenic activities of GBP-1 have not been elucidated.

Materials and methods: Comparative transcriptome analysis were performed with human umbilical vein endothelial cells (HUVEC) stably expressing GBP-1 and control cells using HG-U133 Plus 2.0 GenChip (Affymetrix). Results were confirmed at RNA level by RNase protection assay and at protein level by FACS analysis using a monoclonal antibody against ITGA4. Specific knock-down of GBP-1 was done using vector based RNAi technology (shRNA vector systems). Cell spreading and migratory behaviour (wound healing assay) of the HUVEC was studied on fibronectin coated plates.

Results: Comparative transcriptome analysis of HUVEC showed that GBP-1 induces expression of integrin alpha4 (ITGA4), both at mRNA and protein level. In addition, treatment of HUVEC with interleukin (IL)-1β and tumor necrosis factor (TNF)-α, both activators of endogenous GBP-1 expression, induced ITGA4 expression, whereas specific knock-down of GBP-1 in these cells abrogated ITGA4 expression. These findings indicated that GBP-1 expression was both, necessary and sufficient for the induction of ITGA4 expression by IL-1β and TNF-α. Further cell biological characterization showed that increased ITGA4 expression inhibited cell spreading and migration of HUVEC on fibronectin and that both features could be restored by inhibition of GBP-1 expression.

Conclusion: Cell spreading and migration are key events during angiogenesis. The inhibition of cell spreading and migration through up-regulation of ITGA4 provides a novel mechanism to explain the anti-invasive activity of GBP-1 in endothelial cells.