Gas6 Stimulates Angiogenesis of Human Retinal Endothelial Cells and of Zebrafish Embryos via ERK1/2 Signaling

Aim: To determine if growth arrest-specific 6 (Gas6) plays an important role in the regulation of angiogenesis in human retinal microvascular endothelial cells (HRMECs) and in vessel development of zebrafish. Methods: Proliferation, wound-healing cell migration, and tube formation were measured in HRMECs treated with recombinant human Gas6 (rhGas6). Sprague-Dawley rat aortas in Matrigels were treated with rhGas6, and microvessel sprouting emanating from arterial rings was analyzed. Transgenic zebrafish embryos (flk:GFP) were microinjected with rhGas6 at 50 hours post-fertilization (hpf), and ectopic sprouting of subintestinal vessels (SIVs) was observed under a confocal microscope. Morpholino oligonucleotides (MOs) were microinjected to knockdown gas6 in zebrafish embryos, and intersegmental vessel impairment was observed. The effect of the extracellular signal-regulated kinase (ERK1/2) inhibitor on the migration of HRMECs and on vessel development in zebrafish embryos was tested. Results: rhGas6 stimulated proliferation, migration, and tube formation in HRMECs in a dose-dependent manner. In rat aortas, rhGas6 induced vessel outgrowth, and the sprouting length was longer than that of controls. The rhGas6-microinjected zebrafish embryos had significantly increased vessel outgrowth in the SIVs. Recombinant human vascular endothelial growth factor (rhVEGF) served as a positive control. Knockdown of gas6 inhibited angiogenesis in the developing vessels of zebrafish. The ERK1/2 inhibitor inhibited HRMEC migration and intersegmental vessel formation in zebrafish embryos. Conclusions/Interpretations: These data suggest that Gas6 plays a pivotal role in proliferation, migration, and sprouting of angiogenic endothelial cells in the retina and in zebrafish embryos. Furthermore, Gas6 induced angiogenic processes are induced via phosphorylation of ERK1/2.