Carboxyethylpyrrole oxidative protein modifications stimulate neovascularization: Implications for age-related macular degeneration

doi:10.1073/pnas.0601552103

Carboxyethylpyrrole oxidative protein modifications stimulate neovascularization: Implications for age-related macular degeneration

*Cole Eye Institute and
^§Lerner Research Institute, Cleveland Clinic Foundation, and
^‡Department of Chemistry, Case Western Reserve University, Cleveland, OH 44195

Edited by Mark T. Keating, Novartis Institutes for Biomedical Research, Cambridge, MA, and approved July 17, 2006
↵ ^†Q.E. and K.R. contributed equally to this work. (received for review February 27, 2006)

Abstract

Choroidal neovascularization (CNV), the advanced stage of age-related macular degeneration (AMD), accounts for >80% of vision loss in AMD. Carboxyethylpyrrole (CEP) protein modifications, uniquely generated from oxidation of docosahexaenoate-containing lipids, are more abundant in Bruch’s membrane from AMD eyes. We tested the hypothesis that CEP protein adducts stimulate angiogenesis and possibly contribute to CNV in AMD. Human serum albumin (HSA) or acetyl-Gly-Lys-O-methyl ester (dipeptide) were chemically modified to yield CEP-modified HSA (CEP-HSA) or CEP-dipeptide. The in vivo angiogenic properties of CEP-HSA and CEP-dipeptide were demonstrated by using the chick chorioallantoic membrane and rat corneal micropocket assays. Low picomole amounts of CEP-HSA and CEP-dipeptide stimulated neovascularization. Monoclonal anti-CEP antibody neutralized limbal vessel growth stimulated by CEP-HSA, whereas anti-VEGF antibody was found to only partially neutralize vessel growth. Subretinal injections of CEP-modified mouse serum albumin exacerbated laser-induced CNV in mice. In vitro treatments of human retinal pigment epithelial cells with CEP-dipeptide or CEP-HSA did not induce increased VEGF secretion. Overall, these results suggest that CEP-induced angiogenesis utilizes VEGF-independent pathways and that anti-CEP therapeutic modalities might be of value in limiting CNV in AMD.

oxidation

Footnotes

^¶To whom correspondence may be addressed at:
Cole Eye Institute (i31), Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195.
E-mail: crabbj{at}ccf.org
^‖To whom correspondence may be addressed at:
Cole Eye Institute (i3-161), Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195.
E-mail: anandab{at}ccf.org
Author contributions: Q.E., J.W.C., and B.A.-A. designed research; Q.E., K.R., J.S., and L.L. performed research; X.G. and R.G.S. contributed new reagents/analytic tools; Q.E., K.R., A.V., J.W.C., and B.A.-A. analyzed data; and J.W.C. and B.A.-A. wrote the paper.
Conflict of interest statement: J.W.C. is a consultant for Alcon Research Ltd. and obtains research funding from Merck & Co. and Johnson & Johnson. J.W.C. and R.G.S. each have a license for CEP as an inventor from Franz Biomarkers LLC.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

AMD,

age-related macular degeneration;

CEP,

2-(ω-carboxyethyl)pyrrole;

CEP-dipeptide,

CEP-modified acetyl-Gly-Lys-O-methyl ester;

HSA,

human serum albumin;

CEP-HSA,

CEP-modified HSA;

MSA,

mouse serum albumin;

CEP-MSA,

CEP-modified MSA;

CNV,

choroidal neovascularization;

CAM,

chick chorioallantoic membrane;

DHA,

docosahexaenoate;

PUFA,

polyunsaturated fatty acid;

RPE,

retinal pigment epithelium.