A phage display-based approach to investigate abnormal neovessels of the retina.
OBJECTIVE
The goal of this project was to identify markers of abnormal neovascularization of the retinal vasculature, which is quintessential of pathologic angiogenesis that occurs in blinding diseases such as proliferative diabetic retinopathy.


METHODS
Abnormal retinal neovascularization was induced in rat pups by subjecting them to the 50/10 oxygen-induced retinopathy (OIR) protocol, which involves fluctuating levels of ambient oxygen. A peptide library (that was displayed on phage) was positively and negatively screened over the surface of retinas isolated from experimental and control rats, respectively. Binding of phage to retinal vessels was evaluated by confocal microscopy of retinal flat mounts decorated with fluorescently labeled phage. The topography of the inner limiting membrane was studied by scanning electron microscopy.


RESULTS
Screening a library of peptides displayed on phage over the surface of OIR retinas resulted in isolation of a particular phage (SH phage) that distinguished between abnormal neovessels and the normal vasculature. As expected, the recognition of abnormal neovessels relied on the unique peptide insert of SH phage. Abnormal neovessels consisted of at least three cell types that were present in the following order of descending abundance: endothelial > pericytes > macrophage/microglia. SH phage recognized both endothelial cells and macrophage/microglia. Finally, SH phage decorated abnormal neovessels at an early stage of their genesis.


CONCLUSIONS
Abnormal development of neovessels is associated with early expression of distinct epitopes on the surface of cells within the pathologic vasculature. Screening phage display libraries is one approach to detecting such changes, and the resulting phage are potential imaging tools and/or drug delivery vehicles.
Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts 02114, USA.
Magdalena
M
Staniszewska
Xiaolin
X
Gu
Carmelo
C
Romano
Andrius
A
Kazlauskas
D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000378:metabolism*; D004195:Disease Models, Animal; D005136:Eye Proteins / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D010100:Oxygen / Q000633:toxicity; D010446:Peptide Fragments / Q000378:metabolism*; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D015861:Retinal Neovascularization / Q000378:metabolism* / Q000473:pathology; D012171:Retinal Vessels / Q000378:metabolism* / Q000648:ultrastructure
D015415:Biomarkers; D005136:Eye Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D010100:Oxygen