Abstract 10707: A Novel Nucleic Acid Analogue 2-Cl-C.OXT-A Promotes Angiogenesis Via Suramin-sensitive Purinergic Receptors and Src Family Tyrosine Kinases (SFK)
We have recently developed a novel nucleic acid analogue structurally resembling adenosine, termed 2-Cl-C.OXT-A (figure), that elicits strong angiogenic activities both in vitro and in vivo via MAP kinases ERK1/2 (BBRC 2010, 399, 699). We explored how 2-Cl-C.OXT-A modulates cellular signaling machineries to promote angiogenesis using cultured human umbilical vein endothelial cells (HUVEC) as a model. Phospho-western analyses revealed that 2-Cl-C.OXT-A leads to robust activation of MAP kinases ERK1/2 in a time-and dose-dependent fashion, assessed as the degrees of ERK1/2 T202/Y204 phosphorylation (13.7+3.5 fold over basal, p<0.05 at 100 microM for 20 min), associated with activation of up-stream MAP kinase kinase MEK1/2 as well as Raf-1 MAP kinase kinase kinase. ERK1/2 activation by 2-Cl-C.OXT-A was sensitive to suramin (100 microM for 30 min), an inhibitor of P2 family G-protein coupled purinergic receptors; BAPTA-AM (20 microM for 30 min), an intracellular calcium chelator; and U73112 (5 microM for 30 min), an inhibitor of phospholipase C (PLC). Immunoprecipitation assay followed by phospho-western analyses using the PY20 antibody showed that 2-Cl-C.OXT-A (100 microM for 20 min) induces remarkable phosphorylation of p130CAS (2.5+0.2 fold over basal, p<0.05 at 100 microM for 20 min), a major substrate protein of SFK, which is abrogated by PP2 (100 microM for 30 min), a pharmacological SFK inhibitor. Conversely, PP2 significantly attenuated 2-Cl-C.OXT-A-elicited promotion of both ERK1/2 activation (phospho-western assay) and tube formation activity (CD-31 staining). Collectively, these results are consistent with a model in which 2-Cl-C.OXT-A exerts its angiogenic activity by way of P2 family purinergic receptors to activate PLC and mobilize intracellular calcium, leading to activation of a protein kinase cascade comprising Src-family tyrosine kinases and MAP kinases, thus identifying a novel endothelial machinery regulating angiogenesis.
- © 2012 by American Heart Association, Inc.