Abstract
Purpose:
The normal cornea is an avascular tissue. However, the mechanisms of avoiding blood vessels into cornea have not been fully understood yet. To identify the key molecules for keeping cornea avascular, we previously compared the expression gene profile between cultured human dermal fibroblasts (HDFs) and keratocytes(HKCs) using microarray analysis and identified several candidate genes. In this study, the anti-angiogenic features of one of these genes (angiopoietin like protein 7; angptl7) were examined in vitro and in vivo.
Methods:
Random sequence and angptl7 siRNAs were transfected to HKCs. Human umbilical vein endothelial cells (HUVECs) were cultured on HKCs for 6 days with or without recombinant human Angptl7. The tube formation was examined by immunohistochemistry using anti-CD31 antibody, and the areas of CD31-positive tube formation were quantified by ImageJ software. Random sequence and angptl7 PshRNAs (PshRNA is a new class of RNAi agent which is synthesized singe strand RNA to avoid immune-mediated toxicities of siRNA.) were transfected to corneal stroma of C57BL/6. Day6 after injection, neovuscularization areas in these corneas were evaluated by immunohistochemistry using anti-CD31 antibody, and the areas of neovuscularization were quantified by ImageJ software.
Results:
The mRNA expression level of angptl7 was significantly inhibited by angptl7 siRNA transfection compared to random sequence siRNA transfection. The tube formation areas in angptl7 siRNA treatment significantly compared to that in random siRNA treatment. The recombinant angptl7 administration abolished the tube formation by angptl7 siRNA treatment. The neovuscularization areas of mice corneas after transfected Angptl7 PshRNA were significantly higher than those transfected random sequence PshRNA.
Conclusions:
This study revealed that Angptl7 acts as an anti-angiogenic factor in corneal angiogenesis in vitro and in vivo. Angptl7 expressed abundantly in keratocytes, may be an important molecule to keep cornea avascular.
Keywords: 480 cornea: basic science •
484 cornea: stroma and keratocytes •
609 neovascularization