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C.M. Bojanowski, D. Shen, R.J. Ross, E.Y. Chew, W.R. Green, C.–C. Chan, J. Tuo; Variation Within Apolipoprotein E May Protect Against Age–Related Macular Degeneration Through Cytokine Regulation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4156.
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CCL2 (a chemokine) and VEGF (a cytokine) have previously been associated with age–related macular degeneration (AMD). Early AMD is characterized by an accumulation of lipid–containing drusen within Bruch’s membrane. ApoE, a major polymorphic CNS apolipoprotein, is found in high levels in soft drusen as well as in eyes affected by neuroretinal damage. Variation within ApoE (at positions C112R and R158C) is associated with AMD. The aim of this study is to determine the possible mechanisms underlying this association by studying the effects of recombinant ApoE isoforms on the expression of CCL2 and VEGF in cultured retinal pigmented epithelium (RPE) cells.
Genomic DNA was PCR amplified following extraction from peripheral blood collected from 299 age–matched controls with no clinical signs of AMD and 407 advanced AMD patients from the AREDS cohort. Genomic DNA was also collected, following microdissection and whole genome amplification, from 40 archived paraffin–embedded ocular slides of pathologically diagnosed AMD patients. ApoE Cys112Arg (rs#429358) and Arg158Cys (rs#7412) SNP typing was performed using both RFLP and Taqman allele discrimination assays. Functional studies conducted by co–incubation of RPE cells with the ApoE E3 and E4 isoforms were performed. Culture mediums were subjected to ELISA assays for CCL2 and VEGF analysis.
ApoE 112R distribution differed significantly between AMD patients and controls with allele frequencies of 5% (4/80) in the archived AMD cases, 10.7% (87/814) in AMD patients, and 14.6% (87/598) in the age–matched controls. Significant differences in ApoE 158C distribution were not observed. In vitro studies found that recombinant ApoE suppresses CCL2 and VEGF expression in RPE cells. However, the E4 isoform (112R/158R) showed greater suppression than E3 (112C/158R) in both cases.
The results of our study further confirm the association between the ApoE 112R non–synonymous variant and a decreased risk of AMD development. In vitro studies suggest that one of the underlying mechanisms of this association may involve the differential regulation of both CCL2 and VEGF expression in RPE cells by the ApoE isoforms. This finding may help to identify targeted therapeutic strategies for the treatment of this debilitating disease.
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