Abstract
Abstract: :
Purpose:1) To develop a reliable murine model of exudative AMD by combining the effects of three major risk factors for the disease: age, diet and apolipoprotein E (APOE) isoform expression. 2) To characterize the disease phenotype. 3) To identify growth factors commonly expressed in choroidal neovascularization (CNV) in this model. Methods: Three human APOE targeted replacement (TR) mouse lines, each expressing one of the three common human APOE isoforms (ε2, ε3, ε4), were created by replacing the coding sequences of mouse apoE with human APOE allele–specific coding sequences, without disturbing murine regulatory sequences. Aged mice (average 103 weeks) were bred and housed conventionally, and fed a sodium cholate diet for 8 weeks. Mice on normal chow served as controls. Blood was collected, and serum cholesterol levels were measured. For light microscopy, eyes were fixed in paraformaldehyde and cryoprotected. For electron microscopy eyes were fixed in gluteraldehyde and embedded in Spurr resin. For protein detection, eyes were frozen in liquid nitrogen and homogenized in 2% SDS. Proteins assayed included apoE, VEGF, FGF, TGFß, and IGF–1. Results: APOE TR mice express APOE isoforms at physiological levels in patterns similar to wildtype mice and humans. Serum cholesterol levels in all genotypes of cholate–fed animals were greater than animals on normal diet. Histologically, retinas of APOE mice on normal chow were unremarkable. APOE2 mice on the cholate diet showed vacuolization, sub–retinal pigment epithelial (sub–RPE) deposits and photoreceptor and RPE loss. APOE3 retinas had less severe RPE phenotype with fewer vacuoles. Only the APOE4 retinas showed VEGF positive neovascularization as well as RPE changes including atrophy. Total apoE protein levels in APOE2 mice increased following cholate diet but remained the same in APOE3 and E4 animals. In mice on the normal diet, apoE levels varied as follows: APOE2<APOE3<APOE4. Conclusions: Our findings suggest that specific APOE isoform expression alone results in changes in retinal integrity. However, the APOE2 and especially APOE4 isoforms, when combined with age and a diet rich in cholate (which enhances cholesterol absorption), increase degenerative changes in the retina/choroid. Therefore APOE may be a susceptibility gene which confers an increased risk but does not directly cause AMD. These mice provide a reliable animal model for studying factors that may initiate CNV.
Keywords: age–related macular degeneration • choroid: neovascularization • growth factors/growth factor receptors