Gas6 stimulates angiogenesis of human retinal endothelial cells and of zebrafish embryos via ERK1/2 signaling

PLoS One. 2014 Jan 7;9(1):e83901. doi: 10.1371/journal.pone.0083901. eCollection 2014.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aorta / drug effects
  • Aorta / metabolism
  • Cell Movement / drug effects
  • Cell Movement / genetics
  • Cell Proliferation / drug effects
  • Endothelial Cells / drug effects*
  • Endothelial Cells / metabolism*
  • Gene Knockdown Techniques
  • Gene Order
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Intercellular Signaling Peptides and Proteins / genetics*
  • Intercellular Signaling Peptides and Proteins / pharmacology
  • MAP Kinase Signaling System / drug effects
  • Neovascularization, Physiologic / drug effects
  • Neovascularization, Physiologic / genetics*
  • Phenotype
  • Protein Kinase Inhibitors / pharmacology
  • Rats
  • Retina / metabolism*
  • Zebrafish

Substances

  • Intercellular Signaling Peptides and Proteins
  • Protein Kinase Inhibitors
  • growth arrest-specific protein 6

Grants and funding

This research was supported by grants [K12040 and K13040] from the Korea Institute of Oriental Medicine (KIOM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.