Our results provide further evidence that ApoE112R is associated with a reduced risk of AMD development. Although there are a few negative reports, this association has been previously reported. In the present study, a quantitative negative correlation between ApoE112R and AMD prevalence was observed with a lower OR for the PD AMD cases than for the CD AMD cases when comparing with the same control group. Furthermore, a higher OR was observed for the CD AMD group when compared with the BD control group than when compared with the screened controls. These results suggest a correlation between allele frequency and disease incidence.
In contrast to the protective role of ApoE E4 for AMD, the E4 isoform is associated with an increased risk for AD and various cardiovascular diseases [Mahley and Huang, 1999
; Laws et al., 2003
]. Current knowledge of the differential functioning among the ApoE isoforms, either in lipid metabolism or neuronal protection, does not readily provide a rationale for the observed protective role of E4 against AMD development [Nathan et al., 1994
; Ji et al., 2002
; Laws et al., 2003
]. ApoE is expressed in photoreceptor outer-segments, the retinal ganglion layer, and both layers of Bruch's membrane. ApoE is also basally secreted by RPE cells [Anderson et al., 2001
; Ishida et al., 2004
]. It has been postulated that ApoE may facilitate lipid efflux from RPE cells and lipid transport across Bruch's membrane, thus reducing lipid deposit levels [Souied et al., 1998
]. However, this hypothesis does not account for the fact that ApoE is a significant component of drusen [Anderson et al., 2001
Altered ApoE function caused by the 112R
variant may indeed play a role in AMD pathogenesis. Incubation with wild-type ApoE E3 or variant ApoE E4-enriched 3-VLDL results in differential cellular accumulation [Ji et al., 1998
; Laws et al., 2003
]. A two- to threefold increase in the accumulation of ApoE E3 over ApoE E4 has been observed in various cell types [Ji et al., 2002
]. If mechanisms for proper clearance in the retina and Bruch's membrane are not in place, this accumulation may serve as a pathogenic basis for disease development [Ambati et al., 2003
; Tuo et al., 2004b
]. In fact, the increased accumulation in non-E4 carriers may explain why ApoE is such a ubiquitous component of ocular drusen in AMD-afflicted eyes [Crabb et al., 2002
ApoE also may be involved in AMD pathogenesis through its association with C-reactive protein (CRP) levels. A chronic inflammation theory has been proposed to account for macular drusen etiology [Anderson et al., 2002
]. CRP, a systemic inflammatory marker, is a prominent drusen-associated molecule and is expressed in significantly higher levels among advanced AMD patients [Seddon et al., 2004
]. Persons with one or two copies of ApoE
E4 are associated with reduced CRP levels when compared to individuals lacking these copies [Austin et al., 2004
; Judson et al., 2004
; Marz et al., 2004
]. This finding suggests that an ApoE isoform-mediated inflammatory response may be involved in AMD etiology.
It has been reported that up-regulated macrophage and inflammatory cell function may worsen or exacerbate already existing inflammatory lesions [Espinosa-Heidmann et al., 2003
]. This up-regulation can be spurred by both CCL2 and VEGF expression. Our functional study demonstrated a stronger suppression of both CCL2 and VEGF expression in RPE cells by the ApoE E4 isoform (Figs. and ). These findings implicate a possible mechanism underlying the observed protective effect of this allele. RPE cells constitutively express CCL2, a CC chemokine, and secrete VEGF, a growth factor and cytokine [Holtkamp et al., 1999
; Crane et al., 2000
]. Increased levels of both CCL2 and VEGF in RPE cells are related to oxidative stress and hypoxia within the eye [Spilsbury et al., 2000
; Uetama et al., 2003
]. VEGF is a potent endothelial cell mitogen and angiogenic growth factor strongly expressed in postmortem AMD eyes both with and without choroidal neovascularization [Lip et al., 2001
]. Over-expression of VEGF in the RPE, even if only temporarily, is sufficient to induce choroidal neovascularization in the rat eye [Kliffen et al., 1997
; Spilsbury et al., 2000
]. CCL2 also has been associated with further deterioration and degeneration, especially in advanced disease stages [Ambati et al., 2003
]. Considering that our data show nearly a twofold greater suppression of CCL2 mRNA and protein expression by E4 in RPE cells with similar findings for VEGF, we suggest that investigating the varied effects of the ApoE isoforms on chemokine and cytokine regulation may offer insights into the mechanisms responsible for the observed protective role of the E4 isoform.
As mentioned previously, the ApoE E4 isoform has been correlated with elevated total cholesterol levels [Smith, 2000
; Ribalta et al., 2003
]. This association remains true in this study (Tables and ). Although it is difficult to draw any mechanistic conclusions from this correlation, these results do support the validity and accuracy of our SNP typing methodology. These results also suggest a serum cholesterol-independent disease pathway in AMD [van Leeuwen et al., 2004
Although difficulty was met in recruiting age-matched controls for an older AMD group, the likelihood of a false-positive was not increased. The incorporation of more closely age-matched controls would certainly increase the statistical power of this study. It is possible that some of the individuals in the BD control group will develop AMD later in life due to the age-related nature of the disease. Therefore, if the 112R polymorphism is a true protective factor against AMD, an even greater difference in the polymorphic frequencies between the case and control groups is anticipated if a more closely age-matched control population was included. This may explain why a lower OR was obtained for the CD AMD group when compared to the BD controls. With the lowest OR of 112R carriers in the PD AMD group, our results indicate that a clearly-defined phenotype also will increase the likelihood of successful genetic factor discrimination.
In conclusion, this study confirms that ApoE112R is associated with a deceased risk of AMD development. The underlying mechanisms of AMD development may involve altered regulation of CCL2 and VEGF expression in RPE cells by the ApoE isoforms. We are continuing to investigate the potential interaction among ApoE, other AMD-associated genes, and environmental factors.