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Polo-like kinase 2 regulates angiogenic sprouting and blood vessel development.

Developmental biology (2015-05-26)
Hongbo Yang, Longhou Fang, Rui Zhan, Jeffrey M Hegarty, Jie Ren, Tzung K Hsiai, Joseph G Gleeson, Yury I Miller, JoAnn Trejo, Neil C Chi
ABSTRACT

Angiogenesis relies on specialized endothelial tip cells to extend toward guidance cues in order to direct growing blood vessels. Although many of the signaling pathways that control this directional endothelial sprouting are well known, the specific cellular mechanisms that mediate this process remain to be fully elucidated. Here, we show that Polo-like kinase 2 (PLK2) regulates Rap1 activity to guide endothelial tip cell lamellipodia formation and subsequent angiogenic sprouting. Using a combination of high-resolution in vivo imaging of zebrafish vascular development and a human umbilical vein endothelial cell (HUVEC) in vitro cell culture system, we observed that loss of PLK2 function resulted in a reduction in endothelial cell sprouting and migration, whereas overexpression of PLK2 promoted angiogenesis. Furthermore, we discovered that PLK2 may control angiogenic sprouting by binding to PDZ-GEF to regulate RAP1 activity during endothelial cell lamellipodia formation and extracellular matrix attachment. Consistent with these findings, constitutively active RAP1 could rescue the endothelial cell sprouting defects observed in zebrafish and HUVEC PLK2 knockdowns. Overall, these findings reveal a conserved PLK2-RAP1 pathway that is crucial to regulate endothelial tip cell behavior in order to ensure proper vascular development and patterning in vertebrates.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Rap1 Activation Assay Kit, Non-radioactive Rap1 Activation Assay Kit that uses Ral GDS RBD, agarose (Catalog # 14-455) to precipitate Rap1-GTP from cell lysates & detection by a Rap1 specific antibody.
Roche
Protein A Agarose, >98% (HPLC and SDS-PAGE), suspension
Sigma-Aldrich
Anti-Integrin αVβ3 Antibody, clone LM609, clone LM609, Chemicon®, from mouse
Sigma-Aldrich
Monoclonal Anti-RAPGEF2 antibody produced in mouse, clone 1E8, purified immunoglobulin, buffered aqueous solution