Purpose.: The authors conducted an in vitro investigation of the role of Ca 2+-dependent signaling in vascular endothelial growth factor (VEGF)-induced angiogenesis in the retina.

Methods.: Bovine retinal endothelial cells (BRECs) were stimulated with VEGF in the presence or absence of 1, 2-bis (2-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; intracellular Ca 2+ chelator), U73122 (phospholipase C (PLC) inhibitor), xestospongin C (Xe-C), and 2-aminoethoxydiphenyl borate (2APB)(inhibitors of inositol-1, 4, 5 triphosphate (IP 3) signaling). Intracellular Ca 2+ concentration ([Ca 2+] i) was estimated using fura-2 Ca 2+ microfluorometry, Akt phosphorylation quantified by Western blot analysis, and angiogenic responses assessed using cell migration, proliferation, tubulogenesis, and sprout formation assays. The effects of the Ca 2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 were also evaluated on VEGF-induced Akt signaling and angiogenic activity.

Results.: Stimulation of BRECs with 25 ng/mL VEGF induced a biphasic increase in [Ca 2+] i, with an initial transient peak followed by a sustained plateau phase. VEGF-induced [Ca 2+] i increases were almost completely abolished by pretreating the cells with BAPTA-AM, U73122, Xe-C, or 2APB. These agents also inhibited VEGF-induced phosphorylation of Akt, cell migration, proliferation, tubulogenesis, and sprouting angiogenesis. KN93 was similarly effective at blocking the VEGF-induced activation of Akt and angiogenic responses.

Conclusions.: VEGF increases [Ca 2+] i in BRECs through activation of the PLC-IP 3 signal transduction pathway. VEGF-induced phosphorylation of the proangiogenic protein Akt is critically dependent on this increase in [Ca 2+] i and the subsequent activation of CaMKII. Pharmacologic inhibition of Ca 2+-mediated signaling in retinal endothelial cells blocks VEGF-induced angiogenic responses. These results suggest that the PLC/IP 3/Ca 2+/CaMKII signaling pathway may be a rational target for the treatment of angiogenesis-related disorders of the eye.