Purpose: Human amniotic membrane (HAM) transplantation is commonly used in corneal surface reconstruction and is known to inhibit neovascularization of this tissue. The purpose of the present study is to reveal the molecular basis underlying antiangiogenic activity of HAM.
Methods: The effects of HAM protein on proliferation of vascular endothelial cells and corneal epithelial cells were determined by quantifying viable cells using the MTT assay. The presence of pigment epithelium-derived factor (PEDF) in HAM was demonstrated at the protein level by Western blot analysis and immunohistochemistry using a monoclonal antibody specific to human PEDF. The PEDF concentration was measured by a specific ELISA. The expression of PEDF in HAM was confirmed at the RNA level by RT-PCR and DNA sequencing.
Results: Soluble proteins from HAM inhibited proliferation of human umbilical vein endothelial cells and bovine retinal capillary endothelial cells (BRCECs) while promoting proliferation of bovine cornea epithelial cells. Moreover, the HAM-induced inhibition of BRCECs was neutralized by a specific anti-PEDF antibody. PEDF protein was identified with an abundance of 103.84 +/- 33.21 ng/mg of soluble proteins, which is comparable to that in the retina, a PEDF-rich tissue. PEDF expression was predominantly localized in the basement membrane of HAM. RT-PCR using specific PEDF primers amplified a single product from HAM RNA. The PCR product has a sequence identical with that of human PEDF.
Conclusion: HAM specifically inhibits endothelial cell growth and thus suppresses neovascularization in the cornea. PEDF in HAM has a major role in eliciting this antiangiogenic activity.