Involvement Of S1p(1) Receptor Pathway In Angiogenic Effects Of A Novel Adenosine-Like Nucleic Acid Analog Coa-Cl In Cultured Human Vascular Endothelial Cells

COA-Cl (2Cl-C. OXT-A) is a recently developed adenosine-like nucleic acid analog that promotes angiogenesis via the mitogen-activated protein (MAP) kinases ERK1/2. Endothelial S1P(1) receptor plays indispensable roles in developmental angiogenesis. In this study, we examined the functions of S1P(1) in COA-Clinduced angiogenic responses. Antagonists for S1P(1), W146, and VPC23019, substantially but still partly inhibited the effects of COA-Cl with regard to ERK1/2 activation and tube formation in cultured human umbilical vein endothelial cells (HUVEC). Antagonists for adenosine A1 receptor and purinergic P2Y(1) receptor were without effect. Genetic knockdown of S1P(1) with siRNA, but not that of S1P(3), attenuated COA-Cl-elicited ERK1/2 responses. The signaling properties of COA-Cl showed significant similarities to those of sphingosine 1-phosphate, an endogenous S1P(1) ligand, in that both induced responses sensitive to pertussis toxin (G alpha i/o inhibitor), 1,2-bis(2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), (calcium chelator), and PP2 (c-Src tyrosine kinase inhibitor). COA-Cl elevated intracellular Ca2+ concentration and induced tyrosine phosphorylation of p130Cas, a substrate of c-Src, in HUVEC. COA-Cl displaced [H-3] S1P in a radioligand-binding competition assay in chem-1 cells overexpressing S1P(1). However, COA-Cl activated ERK1/2 in CHO-K1 cells that lack functional S1P(1) receptor, suggesting the presence of additional yet-to-be-defined COA-Cl target in these cells. The results thus suggest the major contribution of S1P(1) in the angiogenic effects of COA-Cl. However, other mechanism such as that seen in CHO-K1 cells may also be partly involved. Collectively, these findings may lead to refinement of the design of this nucleic acid analog and ultimately to development of small molecule-based therapeutic angiogenesis.