Molecular Cloning, Expression and Characterization of a Novel Angiopoietin-Related Protein, ARP4

Abstract

Abstract: : Purpose: Angiopoietin family plays important roles in angiogenesis in pathophysiological conditions. We have identified angiopoietin-related protein 4 (ARP4) as a novel angiopoietin-related factor from human placenta cDNA library. The purpose of this study is to examine the localization and biological characterization of ARP4 in the mouse eye. Methods: We employed a structural and profile-based cloning strategy to isolate a human ARP4 cDNA. ARP4 mRNA expression in mouse eye was estimated by RT-PCR, and localization of ARP4 protein in mouse eye was examined by immunohistochemical analysis with an anti-mouse ARP4 polyclonal antibody. We purified recombinant ARP4 protein to examine the biological function of ARP4 in the endothelial cells. Results: We determined the sequence of human and mouse ARP4. The open reading frames of human and mouse ARP4 were 470 amino-acid (aa) and 457aa, respectively. ARP4 contained the coiled-coil domain in the N-terminal portion and fibrinogen-like domain in the C-terminus, both of which were conserved in previously reported members of angiopoietin family. We found ARP4 in the inner plexus layer (IPL) and retinal and choroidal vessels in mouse eye. In vitro functional analyses revealed that ARP4 suppressed vascular endothelial growth factor (VEGF)-induced endothelial cells migration and tube formation. In addition, both VEGF-induced neovascularization and vascular leakiness were significantly suppressed by an addition of ARP4. Conclusion: We identified ARP4 as an anti-angiogenic modulatory factor, suggesting an important role as therapeutic agent in pathological conditions such as diabetic retinopathy.

Keywords: gene/expression • growth factors/growth factor receptors • neovascularization