Effects of human recombinant PEDF protein and PEDF-derived peptide 34-mer on choroidal neovascularization.
OBJECTIVE
Pigment epithelium-derived factor (PEDF) is a serpin with antiangiogenic properties. Previously, the authors showed that PEDF injected into the subconjunctiva reaches the choroid. Here, they examined the effects of PEDF polypeptide fragments on vessel sprouting and on choroidal neovascularization (CNV) after subconjunctival administration.


METHODS
Recombinant human PEDF (rhuPEDF) was cleaved at its serpin-exposed loop by limited chymotrypsin proteolysis. Synthetic PEDF peptides 34-mer (Asp(44)-Asn(77)) and 44-mer (Val(78)-Thr(121)) were used. Ex vivo chick aortic vessel sprouting assays were performed. CNV was induced in rats by laser injury of Bruch's membrane. Daily subconjunctival injections (0.01-10 pmol/d protein) were performed for 5 days starting at day of injury or at the seventh day after injury. New vessel volumes were quantified using optical sections of choroid/RPE flat-mounts labeled with isolectin-Ib4. PEDF distribution was evaluated by immunofluorescence of choroid/RPE/retina cross-sections.


RESULTS
Full-length rhuPEDF, cleaved rhuPEDF, or peptide 34-mer exhibited ex vivo antiangiogenic activity, but peptide 44-mer was inefficient. PEDF immunostaining around CNV lesions diminished after laser injury. Subconjunctival administration of rhuPEDF or 34-mer at 0.1 pmol/d decreased CNV lesion volumes by 52% and 47%, respectively, whereas those of 44-mer were similar to vehicle injections. Doses of 0.1 and 1 pmol/d rhuPEDF decreased fully developed CNV complex volumes by 45% and 50%, respectively, compared with vehicle injections.


CONCLUSIONS
A functional region for the inhibition of vessel sprouting and CNV resides within the 34-mer region of PEDF. Furthermore, subconjunctival administration of optimal range dosages of rhuPEDF or 34-mer can suppress and regress rat CNV lesions, demonstrating that these agents reach the choroid/RPE complex as functionally active molecules.
Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-0608, USA.
Juan
J
Amaral
S Patricia
SP
Becerra
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects; D002642:Chick Embryo; D002829:Choroid / Q000187:drug effects*; D020256:Choroidal Neovascularization / Q000473:pathology / Q000517:prevention & control*; D003228:Conjunctiva; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007267:Injections; D018613:Microscopy, Confocal; D018919:Neovascularization, Physiologic / Q000187:drug effects; D009414:Nerve Growth Factors / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D051381:Rats; D011914:Rats, Inbred BN; D011994:Recombinant Proteins / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000187:drug effects*; D015843:Serpins / Q000494:pharmacology*; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor