Abstract
Purpose: :
Amniotic membrane (AM) stroma is avascular; the mechanism of its avascularity remains elusive. It remains unknown if the putative anti–angiogenic mechanism of AM explains why the ocular surface receiving AM transplantation prevents abnormal blood vessel formation. Besides pigment epithelium–derived factor (PEDF), which is an anti–angiogenic factor found predominantly in the amniotic basement membrane, we speculate that AM stroma must contain additional anti–angiogenic factor(s).
Methods: :
Water–soluble AM stromal extracts (ASE) was harvested from scraped cryopreserved AM stroma by PBS extraction. Cultures of human umbilical cord vascular endothelial cells (HUVEC), human corneal fibroblasts (HCF), and rabbit corneal epithelial cell line (RCE) were added with different concentrations of ASE. Cell proliferation was determined by MTT assay, while cell apoptosis was measured by LIVE/DEAD assay and Hoechst 33342 staining. The effect of ASE on VEGF–induced chemotaxis on HUVEC was tested in Boyden chambers.
Results: :
Migration of HUVEC cells with or without chemotatic VEGF was both significantly inhibited by ASE at the concentration of 200 µg/ml for 4 h. ASE inhibited proliferation of HUVEC cells starting from the concentration of 50 µg/ml, but inhibited proliferation of HCF and RCE only at the concentration of 200 µg/ml for 48 h. HUVEC cells, but not HCF and RCE cells, exhibited significant apoptosis when cultured with 200 µg/ml ASE for 48 h.
Conclusions: :
AM stroma contains water–soluble factor(s) that exert a significant anti–angiogenic action through inhibiting migration and proliferation and inducing apoptosis of HUVEC cells. Biochemical identification of such anti–angiogenic factor(s) is under way.
Keywords: neovascularization • apoptosis/cell death