Suppression of corneal neovascularization by PEDF release from human amniotic membranes.
OBJECTIVE
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.


CONCLUSIONS
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.
Shao
Chunkui
C
Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, USA.
Sima
Jing
J
Zhang
Sarah X
SX
Jin
Ji
J
Reinach
Peter
P
Wang
Zheng
Z
Ma
Jian-xing
JX
D000650:Amnion / Q000378:metabolism*; D020533:Angiogenesis Inhibitors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D002455:Cell Division / Q000187:drug effects; D016510:Corneal Neovascularization / Q000517:prevention & control*; D004730:Endothelium, Vascular / Q000166:cytology; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000166:cytology; D005136:Eye Proteins; D006801:Humans; D007124:Immunoenzyme Techniques; D009414:Nerve Growth Factors; D011506:Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology; D000099174:Pigment Epithelium-Derived Factor
D020533:Angiogenesis Inhibitors; D005136:Eye Proteins; D009414:Nerve Growth Factors; D011506:Proteins; D012333:RNA, Messenger; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor