Abstract 11218: S1P1 Receptors Mediate Angiogenic Responses of a Novel Adenosine Analogue COA-Cl in Human Umbilical Vein Endothelial Cells (HUVEC)
We have recently developed a novel adenosine analogue termed COA-Cl (figure) that elicits strong angiogenic activities comparable to those by VEGF, acting through the classic MAP kinase cascade Raf1-MEK-ERK1/2. We explored molecular target of COA-Cl that transmits extracellular stimulation to intracellular signal transduction. The S1P1 receptor for sphingosine 1-phosphate is a G-protein coupled receptor that is indispensable during normal blood vessel development in utero. Because both COA-Cl and S1P are strongly angiogenic and are small molecule agents, we developed a hypothesis that COA-Cl utilizes S1P1 for its signaling. Western blot analyses (WB) in HUVEC revealed that phosphorylation/activation of MAP kinase ERK1/2 elicited by COA-Cl (6.4±1.4 fold vs vehicle, p<0.01) was markedly abrogated by W146, a specific antagonist for S1P1 by 65±15% (p<0.05 vs COA-Cl alone), and by VPC23019, a dual antagonist for S1P1/S1P3. When endothelial tube forming activity was assessed by means of a co-culture assay of HUVEC with normal human fibroblasts followed by CD31 staining, COA-Cl-induced promotion of tube formation responses were dramatically reduced by W146 and by VPC23019. We further characterized signaling responses of HUVEC to COA-Cl in comparison with those induced by S1P1. ERK1/2 activation by COA-Cl was sensitive to: pertussis toxin, an inhibitor of G-protein α i/o subunits; U73122, an inhibitor of phospholipase-C (PLC); and BAPTA-AM, an intracellular calcium chelator. An immunoprecipitation assay revealed that COA-Cl induced significant tyrosine phosphorylation of p130CAS, a known substrate of Src-family tyrosine kinases (SFK). Conversely, PP2, an inhibitor of SFK, abrogates COA-Cl-induced responses at both levels of ERK1/2 phosphorylation and tube formation. Collectively, our results point out the S1P1 receptor subtype as a target of COA-Cl, at which this adenosine analogue mediates angiogenic responses in human vascular endothelial cells.
- © 2013 by American Heart Association, Inc.