Abstract 16221: Scribble-Mediated Polarity of Endothelial Cells is Required for Angiogenesis
Polarity is a characteristic particular of epithelial cells, which is mediated by several protein complexes, one of them being the Scribble/Dlg/Lgl-module. We hypothesize that polarity proteins are also present in endothelial cells to control directed processes involved in angiogenesis. RT-PCR revealed that Scribble (Scrib), Dlg5 and Lgl1 are expressed in human umbilical vein endothelial cells (HUVEC) and Scrib protein abundance was similar between HUVEC and epithelial cell lines. Intracellular polarization as determined from the position of the nucleus opposite of the leading edge during migration was blocked by RNAi against Scrib. Scrib also mediated the polarization of HUVEC within a chemokine gradient: Scrib RNAi blocked the directed migration in a chemotaxis and a transwell assay, whereas random migration in a scratched wound assay was not affected. Co-immunoprecipitation and protein identification by LTQ-Orbitrap-mass spectroscopy identified an interaction of Scrib with integrin α5 and small GTPases suggesting an orchestration of protein interactions at the leading edge of a migrating cell. Accordingly, endothelial sprouting in the spheroid assay was blocked by Scrib-RNAi whereas in the Matrigel assay tube formation was increased by blockade of Scrib expression. Likewise, in a compensation assay, downregulation of Scrib favoured the positioning of the cell in the stalk cell rather than in the tip cell position. To relate this to in vivo angiogenesis, the fliGFP zebrafish model was used. Scrib morpholinos induced bleedings in the brain and transient formation of arterial-venous shunts in the caudal circulation compatible with a delayed angiogenic development of the fish. In conclusion, the polarity protein Scrib represents a new endothelial protein regulating the angiogenic process in vitro and in vivo. Scrib facilitates the translation of chemokine gradients into directed migration and is therefore required for sprouting angiogenesis.
- © 2010 by American Heart Association, Inc.