Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth.
OBJECTIVE
To determine the effect of pigment epithelium-derived factor (PEDF) in a mouse model of ischemia-induced retinal neovascularization and on vascular endothelial growth factor (VEGF)--induced migration and growth of cultured microvascular endothelial cells.


METHODS
Human recombinant PEDF was expressed in the human embryonic kidney 293 cell line and purified by ammonium sulfate precipitation and cation exchange chromatography. C57BL/6 mice were exposed to 75% oxygen from postnatal day (P)7 to P12 and then returned to room air. Mice received intravitreal injections of 2 microg PEDF in one eye and vehicle in the contralateral eye on P12 and P14. At P17, mice were killed and eyes enucleated for quantitation of retinal neovascularization. The mitogenic and motogeneic effects of VEGF on cultured bovine retinal and adrenal capillary endothelial cells were examined in the presence or absence of PEDF, using cell counts and migration assays.


RESULTS
Two species of human recombinant PEDF, denoted A and B, were purified to apparent homogeneity. PEDF B appeared to comigrate on SDS-PAGE with PEDF from human vitreous samples. Changes in electrophoretic mobility after peptide-N-glycosidase F (PNGase F) digestion suggest that both PEDF forms contain N-linked carbohydrate. Analyses of the intact proteins by liquid chromatography-electrospray mass spectrometry (LC-ESMS) revealed the major molecular weight species for PEDF A (47,705 +/- 4) and B (46,757 +/- 5). LC-ESMS analysis of tryptic peptides indicated that PEDF A and B exhibit differences in glycopeptides containing N-acetylneuraminic acid (NeuAc) and N-acetylhexosamine (HexNAc). Intravitreal administration of either species of PEDF significantly inhibited retinal neovascularization (83% for PEDF A and 55% for PEDF B; P = 0.024 and 0.0026, respectively). PEDF A and B (20 nM) suppressed VEGF-induced retinal microvascular endothelial cell proliferation by 48.8% and 41.4%, respectively, after 5 days (P < 0.001) and VEGF-induced migration by 86.5% +/- 16.7% and 78.1% +/- 22.3%, respectively, after 4 hours (P = 0.004 and P = 0.008, respectively).


CONCLUSIONS
These data indicate that elevated concentrations of PEDF inhibit VEGF-induced retinal endothelial cell growth and migration and retinal neovascularization. These findings suggest that localized administration of PEDF may be an effective approach for the treatment of ischemia-induced retinal neovascular disorders.
Duh
Elia J
EJ
Department of Ophthalmology, Johns Hopkins University School of Medicine, Maumenee 809, 600 N. Wolfe Street, Baltimore, MD 21287, USA.
Yang
Hoseong S
HS
Suzuma
Izumi
I
Miyagi
Masaru
M
Youngman
Elaine
E
Mori
Keisuke
K
Katai
Miyuki
M
Yan
Lin
L
Suzuma
Kiyoshi
K
West
Karen
K
Davarya
Shekar
S
Tong
Patrick
P
Gehlbach
Peter
P
Pearlman
Joel
J
Crabb
John W
JW
Aiello
Lloyd P
LP
Campochiaro
Peter A
PA
Zack
Donald J
DJ
D000818:Animals; D015153:Blotting, Western; D002452:Cell Count; D002455:Cell Division / Q000187:drug effects*; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D016228:Endothelial Growth Factors / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000473:pathology*; D005136:Eye Proteins; D015870:Gene Expression; D006801:Humans; D008222:Lymphokines / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009414:Nerve Growth Factors; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use*; D011994:Recombinant Proteins; D015427:Reperfusion Injury / Q000150:complications / Q000188:drug therapy* / Q000473:pathology; D015861:Retinal Neovascularization / Q000188:drug therapy* / Q000209:etiology / Q000473:pathology; D012171:Retinal Vessels / Q000187:drug effects / Q000473:pathology; D015843:Serpins / Q000235:genetics / Q000302:isolation & purification / Q000627:therapeutic use*; D021241:Spectrometry, Mass, Electrospray Ionization; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000099174:Pigment Epithelium-Derived Factor
D016228:Endothelial Growth Factors; D005136:Eye Proteins; D008222:Lymphokines; D009414:Nerve Growth Factors; D011506:Proteins; D011994:Recombinant Proteins; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000099174:Pigment Epithelium-Derived Factor