Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

further understand the pathogenesis of AMD and test potential treatments for the disease, the search for an animal model of AMD has been extensive and so far unsuccessful. This is not surprising as it is likely that many different combinations of environmental and genetic risk factors may lead to development of this multifactorial disease. Here we present a murine model of AMD developed using factors that are each, individually, associated with an increased risk for the disease.

2. AMD RISK FACTORS

Although the etiology of AMD remains largely unknown, numerous studies have implicated both genetic and environmental influences, which are reflected in the number of risk factors identified to date (Vingerling et al., 1995b). Known risk factors for AMD include: advanced age, environmental factors including cigarette smoking, nutritional factors such as diets high in fat and cholesterol, systemic diseases including hypercholesterolemia and atherosclerosis, gender, ethnicity, and family history (2000; Cho et al., 2001; Christen et al., 1996; Delcourt et al., 2001). There is strong evidence that genetics play an important role in the pathogenesis of AMD (Heiba et al., 1994; Klaver et al., 1998b; Seddon et al., 1997), such that allelic variations in apolipoprotein E (apoE = protein; APOE = gene), fibulin-5 (Stone et al., 2004), and ABCA4 (Allikmets, 2000), have each, to some degree, been associated with increased risk for AMD.

2.1. ApoE and Neurodegenerative Disease

The APOE gene has been identified as a risk factor for AMD in numerous epidemiological studies (Klaver et al., 1998a; Schmidt et al., 2000; Schultz et al., 2003; Simonelli et al., 2001; Souied et al., 1998), as well as studies using APOE knockout mice (Dithmar et al., 2000) and APOE (*)E3-Leiden mice that carry a dysfunctional form of human APOE- E3 (Kliffen et al., 2000). ApoE forms a lipid protein complex and is an important regulator of cholesterol and lipid clearance, transport and distribution (Mahley, 1988; Mahley and Rall, 2000). It is expressed at high levels in the retina, and is involved in processes that have been implicated in the pathological formation of sub-RPE deposits including drusen (Anderson et al., 2001; Klaver et al., 1998a). ApoE protein has been localized in the eye to RPE, Müller cells, Bruch’s membrane and drusen (Hageman et al., 1999; Klaver et al., 1998a) and APOE mRNA is present in the neural retina and RPE/choroid (Hageman et al., 1999). In humans, the APOE gene is polymorphic and encodes three isoforms: E2, E3, and E4, with frequencies of 7%, 77% and 15%, respectively, in the general population (Davignon et al., 1988). Epidemiological studies of the association of APOE allele with AMD have yielded conflicting results regarding the APOE isoform-associated risk, with many studies describing a protective effect in E4 carriers and detrimental effect in E2 carriers (Schmidt et al., 2002; Schmidt et al., 2000) while others have not been able to find this association (Smith et al., 2001). This is in contrast to its role in other neurodegenerative and systemic diseases including Alzheimer’s, atherosclerosis, amyotrophic lateral sclerosis, and stroke in which there is a very strong negative association of the E4 allele with disease (Kalaria, 1997; Lacomblez et al., 2002; Weller and Nicoll, 2003). In Alzheimer’s disease, for example, there is a 90% risk for the disease associated with the expression of the E4 allele (Higgins et al., 1997).

2.2. Animal Models of AMD

To date there is no generally accepted experimental animal model for dry or exudative AMD. Reproducibility of existing models is limited by technical artifacts or because, as in exudative AMD in humans, with the induction of CNV, there is also a concomitant nonspecific, local inflammatory reaction (Mori et al., 2001; Spilsbury et al., 2000). Though previous studies attempting to simulate AMD using transgenic mice have been very useful, many of the resulting animal models were produced by manipulating genes that are not known AMD risk factors. For example, animals expressing mutant forms of a short chain collagen gene, CTRP5 (Hayward et al., 2003), cathepsin D (Rakoczy et al., 2002), or the very low density lipoprotein receptor gene (Heckenlively et al., 2003) develop various aspects of AMD. Transgenic knockouts of genes expressing proteins that are normally present in the retina at low levels [monocyte chemoattractant protein-1 (MCP-1 or Ccl-2) and chemokine receptor-2 (Ccr-2)(Ambati et al., 2003)] have also produced models of AMD. AMD has been modeled using phototoxicity (Cousins et al., 2002), senescence acceleration (Majji et al., 2000), high fat diets (Cousins et al., 2002; Dithmar et al., 2000; Fliesler et al., 2000; Kliffen et al., 2000; Miceli et al., 2000; Ong et al., 2001), in vitro procedures such as laser photocoagulation of Bruch’s membrane in primates (Miller et al., 1986), rats (Dobi et al., 1989; Frank et al., 1989; Hikichi et al., 2002) and mice (Tobe et al., 1998a; Tobe et al., 1998b), elevated levels of bFGF in minipigs (Soubrane et al., 1994) and rabbits (Kimura et al., 1995), implantation of tumors on or in the eye to stimulate angiogenesis (Ambati et al., 2002), and exposure of newborn animals to high oxygen (McLeod et al., 2002). Collectively, none of these models have successfully correlated their histological, morphological and biochemical features to the key features of AMD.

3. DESIGN OF A NEW ANIMAL MODEL

As described above, epidemiological studies have demonstrated an APOE isoformassociated risk for AMD, supporting a role for APOE isoform-specific effects in the pathogenesis of AMD. Therefore, we are studying transgenic mice expressing each of the three major human APOE isoforms in order to rigorously analyze the role of APOE in the development of AMD.

3.1. APOE Targeted-Replacement Mice

To date, transgenic animals made by pronuclear injection of human DNA have been used to study the effect of APOE expression, but this method produces mice with varying levels of transgene expression due to differences in insertion of the transgene constructs within the host genome, and transgene copy number. This, as well as expression of the endogenous mouse ApoE, complicates interpretation of the effect of the foreign APOE (Sullivan et al., 1997). To overcome these two complications we used targeted gene replacement (TR) mice generated by homologous recombination of human APOE coding sequences with the endogenous murine ApoE gene, made by our collaborator Dr. Patrick Sullivan (Sullivan et al., 1997). These mice express one of the three common human APOE isoforms (E2, E3 or E4) at levels that are in the physiological range. Briefly, coding sequences for the mouse ApoE gene were replaced with coding sequences for human APOE2, E3 or E4,

without disturbing any of the known 5¢ or 3¢ murine regulatory sequences. This gene replacement strategy results in animals that express human APOE2, E3 or E4 mRNAs, identical in tissue distribution and levels to that of mouse APOE mRNA in wild type animals (Sullivan et al., 2004; Sullivan et al., 1997). It should be pointed out that in humans the most common APOE allele is E3 which is equivalent to the murine wild type ApoE. On the other hand, 5- 10% of APOE2 humans will develop Type III hyperlipoproteinemia, whereas 100% of APOE2 TR mice on a high fat diet develop Type III hyperlipoproteinemia (Sullivan et al., 1998).

3.2. Experimental Protocol and Diet Paradigm

APOE expression alone is not sufficient to cause neurodegenerative disease in humans, but is instead a risk factor that when combined with other insults, collectively, leads to development of Alzheimer’s disease, stroke and putatively AMD. Therefore, we used two other known risk factors in our animal model to mimic a multifactorial combination that would produce characteristic features of AMD. The factors were: advanced age and a high fat/high cholesterol diet.

Aged male and female (aged 65-127 weeks) APOE TR mice of each isoform (E2, E3, E4) were bred and housed conventionally and fed a high fat/high cholesterol diet rich in cholate (HF-C) and water ad libitum for 8 weeks. Age-matched controls were fed standard rodent chow and water ad libitum. At the end of the eight week diet exposure, mice were euthanized with an overdose of anesthesia, and the eyes were collected for analysis. Histologically, 10 mm thick cryosections and 1 mm plastic sections were examined at the light microscopic level and ultra thin sections were examined using a transmission electron microscope (EM).

4. HISTOLOGICAL CHARACTERIZATION OF AGED APOE TR MICE

4.1. Light Microscopic Evaluation

There were no remarkable abnormalities by light microscopy in the retina, RPE, Bruch’s membrane and choroid of aged APOE TR mice maintained on normal mouse chow diet (Fig. 17.1A). In aged APOE3 TR mice fed the HF-C (TR-ch), there were no detectable retinal changes and only minor RPE changes including RPE vacuolization. This is not surprising since as mentioned before, in humans the APOE3 allele is equivalent to the murine wild type APOE. APOE2 TR-ch mice showed more RPE vacuolization and were further compromised by RPE blebbing and Bruch’s membrane thickening (Fig. 17.1B, arrow), and basal deposit formation, some RPE and photoreceptor degeneration. These changes were also documented, in the APOE4 TR-ch mouse eyes, which showed the most extensive AMDrelated changes overall. Some APOE4 TR-ch eyes also showed ‘drusen-like’ basal deposits (Fig. 17.1C, arrowhead), and neovascularization (NV) of varying severities (Fig. 17.1D). The NV ranged from mild, confined to an area adjacent to the RPE and Bruch’s membrane without any disruption of the neural retina, to more extensive, proliferating through the RPE, through the sub-retinal space and into the neural retina. Though it appears the NV origi-

Figure 17.1. Light micrographs from plastic sections of posterior retinas of APOE TR mice stained with toluidine blue. A. APOE3 TR mouse fed normal mouse chow. B. APOE2 TR mouse on cholate diet with thickening of Bruch’s membrane (arrow). C. APOE4 TR mouse on cholate diet with ‘drusen-like’ basal deposit (arrowhead) and vacuoles (V). D. APOE4 TR mouse on cholate diet with neovascularization spanning the RPE and the overlying neural retinal layers. Magnification 20X. INL = inner nuclear layer, ONL = outer nuclear layer, PR = photoreceptors, RPE = retinal pigment epithelium.

nates from the choroid, the source remains to be conclusively determined using fluorescein angiography and/or further histological analysis of sequential sections through the NV.

4.2. Electron Microscopic Evaluation

EM was used to further evaluate the histological abnormalities documented at the light microscopic level. In all aged APOE TR mice maintained on the normal diet the retina, RPE, and Bruch’s membrane remained essentially normal. EM analysis of RPE vacuoles initially observed by light microscopy, in APOE4 TR-ch mice, revealed the presence of membraneous material (Fig. 17.2A). In the retinas of APOE2 and E4 TR-ch mice RPE basal infoldings were disorganized or absent (Fig. 17.2B). In APOE4 TR-ch mouse eyes there was Bruch’s membrane thickening (Fig. 17.2C), as well as thick basal deposits between RPE and Bruch’s membrane (Fig. 17.2D). In APOE4 TR-ch mice with neovascularization, thin or absent Bruch’s membrane was observed with vessels adjacent to Bruch’s membrane in both the sub-RPE and choroidal regions (not shown).

Figure 17.2. Electron micrographs of the basal RPE/Bruch’s membrane interface at 5000 X magnification of APOE4 TR mice on high fat/high cholesterol cholate diet. A. Normal basal infoldings with vacuoles that contain whorls of membranous material. B. Disorganized RPE basal infoldings. C. Thickened Bruch’s membrane (arrow). D. Thick basal laminar deposit.

5. CONCLUSIONS

Our findings suggest that specific APOE isoform expression alone results in only mild retinal changes with aging. However, in the APOE2 and especially APOE4 expressing animals, the combination of increased age (65-127 weeks) and a high fat diet rich in cholate (which enhances cholesterol absorption), resulted in more extensive degenerative changes in the retina/choroid reminiscent of both dry and wet AMD changes in the human eye. Hallmarks of AMD including thick diffuse sub-RPE deposits (Figs. 17.1B and 17.2D) and drusen like deposits (Fig. 17.1C), thickening of BrM (Fig. 17.2C), patchy regions of RPE atrophy overlying photoreceptor degeneration, and neovascularization (Fig. 17.1D) were detected in these animals. This is the first report of spontaneously occurring neovascularization and retinal/RPE changes in an animal model developed by combining the following known risks associated with AMD: advanced age, environmental risk (high fat, high cholate diet), and genetic risk [APOE isoform expression, using APOE targeted replacement mice (TR)]. We are currently continuing to examine the features of this model more closely and correlate similarities between this model and the human form of AMD, through imaging of the neovascularization using Fluorescein angiography, immunohistochemical localization of hallmark growth factors and proteins found in human CNV and basal deposits/drusen, in the mouse NV and basal deposits.

6. ACKNOWLEDGEMENTS

The authors gratefully acknowledge Drs. Cynthia Toth and Lincoln Johnson for scientific discussions, and valuable input, and the following funding agencies: NEI RO1 EY11286 (CBR), NEI P30 EY05722 (Core), NIA P50 AG05128-20 (DS, GM), Research to Prevent Blindness Career Development Award (CBR), AHAF MDR 2004 (CBR). We also sincerely thank the National Eye Institute and Foundation for Fighting Blindness for the travel award provided to GM to attend this meeting.

7. REFERENCES

No authors listed, 1991. Subfoveal neovascular lesions in age-related macular degeneration. Guidelines for evaluation and treatment in the macular photocoagulation study. Macular Photocoagulation Study Group. Arch Ophthalmol 109:1242-1257.

No authors listed, 2000. Risk factors associated with age-related macular degeneration. A case-control study in the age-related eye disease study: age-related eye disease study report number 3. Age-Related Eye Disease Study Research Group. Ophthalmology 107:2224-2232.

Allikmets, R., 2000. Further evidence for an association of ABCR alleles with age-related macular degeneration. The International ABCR Screening Consortium. Am J Hum Genet 67:487-491.

Ambati, B. K., Joussen, A. M., Ambati, J., Moromizato, Y., Guha, C., Javaherian, K., Gillies, S., O’Reilly, M. S., and Adamis, A. P. 2002. Angiostatin inhibits and regresses corneal neovascularization. Arch Ophthalmol 120:1063-1068.

Ambati, J., Anand, A., Fernandez, S., Sakurai, E., Lynn, B. C., Kuziel, W. A., Rollins, B. J., and Ambati, B. K. 2003. An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice. Nat Med 9:1390-1397.

Anderson, D. H., Ozaki, S., Nealon, M., Neitz, J., Mullins, R. F., Hageman, G. S., and Johnson, L. V. 2001. Local cellular sources of apolipoprotein E in the human retina and retinal pigmented epithelium: implications for the process of drusen formation. Am J Ophthalmol 131:767-781.

Bird, A. C., Bressler, N. M., Bressler, S. B., Chisholm, I. H., Coscas, G., Davis, M. D., de Jong, P. T., Klaver, C. C., Klein, B. E., Klein, R., et al. 1995. An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group.

Surv Ophthalmol 39:367-374.

Cho, E., Hung, S., Willett, W. C., Spiegelman, D., Rimm, E. B., Seddon, J. M., Colditz, G. A., and Hankinson, S. E. 2001. Prospective study of dietary fat and the risk of age-related macular degeneration. Am J Clin Nutr 73:209-218.

Christen, W. G., Glynn, R. J., Manson, J. E., Ajani, U. A., and Buring, J. E. 1996. A prospective study of cigarette smoking and risk of age-related macular degeneration in men. Jama 276:1147-1151.

Cousins, S. W., Espinosa-Heidmann, D. G., Alexandridou, A., Sall, J., Dubovy, S., and Csaky, K. 2002. The role of aging, high fat diet and blue light exposure in an experimental mouse model for basal laminar deposit formation. Exp Eye Res 75:543-553.

Curcio, C. A., and Millican, C. L. 1999. Basal linear deposit and large drusen are specific for early age-related maculopathy. Arch Ophthalmol 117:329-339.

Davignon, J., Gregg, R. E., and Sing, C. F. 1988. Apolipoprotein E polymorphism and atherosclerosis. Arteriosclerosis 8:1-21.

Delcourt, C., Michel, F., Colvez, A., Lacroux, A., Delage, M., and Vernet, M. H. 2001. Associations of cardiovascular disease and its risk factors with age-related macular degeneration: the POLA study. Ophthalmic Epidemiol 8:237-249.

Dithmar, S., Curcio, C. A., Le, N. A., Brown, S., and Grossniklaus, H. E. 2000. Ultrastructural changes in Bruch’s membrane of apolipoprotein E-deficient mice. Invest Ophthalmol Vis Sci 41:2035-2042.

Dobi, E. T., Puliafito, C. A., and Destro, M. 1989. A new model of experimental choroidal neovascularization in the rat. Arch Ophthalmol 107:264-269.

Fliesler, S. J., Richards, M. J., Miller, C., Peachey, N. S., and Cenedella, R. J. 2000. Retinal structure and function in an animal model that replicates the biochemical hallmarks of desmosterolosis. Neurochem Res 25:685694.

Frank, R. N., Das, A., and Weber, M. L. 1989. A model of subretinal neovascularization in the pigmented rat. Curr Eye Res 8:239-247.

Green, W. R. 1999. Histopathology of age-related macular degeneration. Mol Vis 5:27.

Grossniklaus, H. E., and Green, W. R. 2004. Choroidal neovascularization. Am J Ophthalmol 137:496-503. Hageman, G. S., Mullins, R. F., Russell, S. R., Johnson, L. V., and Anderson, D. H. 1999. Vitronectin is a con-

stituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells. Faseb J 13:477-484.

Hayward, C., Shu, X., Cideciyan, A. V., Lennon, A., Barran, P., Zareparsi, S., Sawyer, L., Hendry, G., Dhillon, B., Milam, A. H., et al. 2003. Mutation in a short-chain collagen gene, CTRP5, results in extracellular deposit formation in late-onset retinal degeneration: a genetic model for age-related macular degeneration. Hum Mol Genet 12:2657-2667.

116 G. MALEK ET AL.

Heckenlively, J. R., Hawes, N. L., Friedlander, M., Nusinowitz, S., Hurd, R., Davisson, M., and Chang, B. 2003. Mouse model of subretinal neovascularization with choroidal anastomosis. Retina 23:518-522.

Heiba, I. M., Elston, R. C., Klein, B. E., and Klein, R. 1994. Sibling correlations and segregation analysis of agerelated maculopathy: the Beaver Dam Eye Study. Genet Epidemiol 11:51-67.

Higgins, G. A., Large, C. H., Rupniak, H. T., and Barnes, J. C. 1997. Apolipoprotein E and Alzheimer’s disease: a review of recent studies. Pharmacol Biochem Behav 56:675-685.

Hikichi, T., Mori, F., Sasaki, M., Takamiya, A., Nakamura, M., Shishido, N., Takeda, M., Horikawa, Y., Matsuoka, H., and Yoshida, A. 2002. Inhibitory effect of bucillamine on laser-induced choroidal neovascularization in rats. Curr Eye Res 24:1-5.

Javitt, J. C., Zhou, Z., Maguire, M. G., Fine, S. L., and Willke, R. J. 2003. Incidence of exudative age-related macular degeneration among elderly Americans. Ophthalmology 110:1534-1539.

Kalaria, R. N. 1997. Arteriosclerosis, apolipoprotein E, and Alzheimer’s disease. Lancet 349:1174.

Kimura, H., Sakamoto, T., Hinton, D. R., Spee, C., Ogura, Y., Tabata, Y., Ikada, Y., and Ryan, S. J. 1995. A new model of subretinal neovascularization in the rabbit. Invest Ophthalmol Vis Sci 36:2110-2119.

Klaver, C. C., Kliffen, M., van Duijn, C. M., Hofman, A., Cruts, M., Grobbee, D. E., van Broeckhoven, C., and de Jong, P. T. 1998a. Genetic association of apolipoprotein E with age-related macular degeneration. Am J Hum Genet 63:200-206.

Klaver, C. C., Wolfs, R. C., Assink, J. J., van Duijn, C. M., Hofman, A., and de Jong, P. T. 1998b. Genetic risk of age-related maculopathy. Population-based familial aggregation study. Arch Ophthalmol 116:1646-1651.

Kliffen, M., Lutgens, E., Daemen, M. J., de Muinck, E. D., Mooy, C. M., and de Jong, P. T. 2000. The (APO*)E3Leiden mouse as an animal model for basal laminar deposit. Br J Ophthalmol 84, 1415-1419.

Lacomblez, L., Doppler, V., Beucler, I., Costes, G., Salachas, F., Raisonnier, A., Le Forestier, N., Pradat, P. F., Bruckert, E., and Meininger, V. 2002. APOE: a potential marker of disease progression in ALS. Neurology 58:1112-1114.

Mahley, R. W. 1988. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 240:622-630.

Mahley, R. W., and Rall, S. C., Jr. 2000. Apolipoprotein E: far more than a lipid transport protein. Annu Rev Genomics Hum Genet 1:507-537.

Majji, A. B., Cao, J., Chang, K. Y., Hayashi, A., Aggarwal, S., Grebe, R. R., and De Juan, E., Jr. 2000. Age-related retinal pigment epithelium and Bruch’s membrane degeneration in senescence-accelerated mouse. Invest Ophthalmol Vis Sci 41:3936-3942.

McLeod, D. S., Taomoto, M., Cao, J., Zhu, Z., Witte, L., and Lutty, G. A. 2002. Localization of VEGF receptor-2 (KDR/Flk-1) and effects of blocking it in oxygen-induced retinopathy. Invest Ophthalmol Vis Sci 43:474482.

Miceli, M. V., Newsome, D. A., Tate, D. J., Jr., and Sarphie, T. G. 2000. Pathologic changes in the retinal pigment epithelium and Bruch’s membrane of fat-fed atherogenic mice. Curr Eye Res 20:8-16.

Miller, H., Miller, B., and Ryan, S. J. 1986. The role of retinal pigment epithelium in the involution of subretinal neovascularization. Invest Ophthalmol Vis Sci 27:1644-1652.

Mitchell, P., Smith, W., and Wang, J. J. 1998. Iris color, skin sun sensitivity, and age-related maculopathy. The Blue Mountains Eye Study. Ophthalmology 105:1359-1363.

Mori, K., Ando, A., Gehlbach, P., Nesbitt, D., Takahashi, K., Goldsteen, D., Penn, M., Chen, C. T., Melia, M., Phipps, S., et al. 2001. Inhibition of choroidal neovascularization by intravenous injection of adenoviral vectors expressing secretable endostatin. Am J Pathol 159:313-320.

Ong, J. M., Zorapapel, N. C., Rich, K. A., Wagstaff, R. E., Lambert, R. W., Rosenberg, S. E., Moghaddas, F., Pirouzmanesh, A., Aoki, A. M., and Kenney, M. C. 2001. Effects of cholesterol and apolipoprotein E on retinal abnormalities in ApoE-deficient mice. Invest Ophthalmol Vis Sci 42:1891-1900.

Rakoczy, P. E., Zhang, D., Robertson, T., Barnett, N. L., Papadimitriou, J., Constable, I. J., and Lai, C. M. 2002. Progressive age-related changes similar to age-related macular degeneration in a transgenic mouse model. Am J Pathol 161:1515-1524.

Sarks, S. H. 1976. Ageing and degeneration in the macular region: a clinico-pathological study. Br J Ophthalmol 60:324-341.

Sarks, S. H., Van Driel, D., Maxwell, L., and Killingsworth, M. 1980. Softening of drusen and subretinal neovascularization. Trans Ophthalmol Soc U K 100:414-422.

Schmidt, S., Klaver, C., Saunders, A., Postel, E., De La Paz, M., Agarwal, A., Small, K., Udar, N., Ong, J., Chalukya, M., et al. 2002. A pooled case-control study of the apolipoprotein E APOE gene in age-related maculopathy.

Ophthalmic Genet 23:209-223.

Schmidt, S., Saunders, A. M., De La Paz, M. A., Postel, E. A., Heinis, R. M., Agarwal, A., Scott, W. K., Gilbert, J. R., McDowell, J. G., Bazyk, A., et al. 2000. Association of the apolipoprotein E gene with age-related macular degeneration: possible effect modification by family history, age, and gender. Mol Vis 6:287-293.

Schultz, D. W., Klein, M. L., Humpert, A., Majewski, J., Schain, M., Weleber, R. G., Ott, J., and Acott, T. S. 2003. Lack of an association of apolipoprotein E gene polymorphisms with familial age-related macular degeneration. Arch Ophthalmol 121:679-683.

Seddon, J. M., Ajani, U. A., and Mitchell, B. D. 1997. Familial aggregation of age-related maculopathy. Am J Ophthalmol 123:199-206.

Simonelli, F., Margaglione, M., Testa, F., Cappucci, G., Manitto, M. P., Brancato, R., and Rinaldi, E. 2001. Apolipoprotein E polymorphisms in age-related macular degeneration in an Italian population. Ophthalmic Res 33:325-328.

Smith, W., Assink, J., Klein, R., Mitchell, P., Klaver, C. C., Klein, B. E., Hofman, A., Jensen, S., Wang, J. J., and de Jong, P. T. 2001. Risk factors for age-related macular degeneration: Pooled findings from three continents.

Ophthalmology 108:697-704.

Soubrane, G., Cohen, S. Y., Delayre, T., Tassin, J., Hartmann, M. P., Coscas, G. J., Courtois, Y., and Jeanny, J. C. 1994. Basic fibroblast growth factor experimentally induced choroidal angiogenesis in the minipig. Curr Eye Res 13:183-195.

Souied, E. H., Benlian, P., Amouyel, P., Feingold, J., Lagarde, J. P., Munnich, A., Kaplan, J., Coscas, G., and Soubrane, G. 1998. The epsilon4 allele of the apolipoprotein E gene as a potential protective factor for exudative age-related macular degeneration. Am J Ophthalmol 125:353-359.

Spilsbury, K., Garrett, K. L., Shen, W. Y., Constable, I. J., and Rakoczy, P. E. 2000. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization. Am J Pathol 157:135-144.

Stone, E. M., Braun, T. A., Russell, S. R., Kuehn, M. H., Lotery, A. J., Moore, P. A., Eastman, C. G., Casavant, T. L., and Sheffield, V. C. 2004. Missense variations in the fibulin 5 gene and age-related macular degeneration. N Engl J Med 351:346-353.

Sullivan, P. M., Mace, B. E., Maeda, N., and Schmechel, D. E. 2004. Marked regional differences of brain human apolipoprotein E expression in targeted replacement mice. Neuroscience 124:725-733.

Sullivan, P. M., Mezdour, H., Aratani, Y., Knouff, C., Najib, J., Reddick, R. L., Quarfordt, S. H., and Maeda, N. 1997. Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis. J Biol Chem 272:17972-17980.

Sullivan, P. M., Mezdour, H., Quarfordt, S. H., and Maeda, N. 1998. Type III hyperlipoproteinemia and spontaneous atherosclerosis in mice resulting from gene replacement of mouse Apoe with human Apoe*2. J Clin Invest 102:130-135.

Tobe, T., Okamoto, N., Vinores, M. A., Derevjanik, N. L., Vinores, S. A., Zack, D. J., and Campochiaro, P. A. 1998a. Evolution of neovascularization in mice with overexpression of vascular endothelial growth factor in photoreceptors. Invest Ophthalmol Vis Sci 39:180-188.

Tobe, T., Ortega, S., Luna, J. D., Ozaki, H., Okamoto, N., Derevjanik, N. L., Vinores, S. A., Basilico, C., and Campochiaro, P. A. 1998b. Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model. Am J Pathol 153:1641-1646.

Vingerling, J. R., Dielemans, I., Hofman, A., Grobbee, D. E., Hijmering, M., Kramer, C. F., and de Jong, P. T. 1995a. The prevalence of age-related maculopathy in the Rotterdam Study. Ophthalmology 102:205-210.

Vingerling, J. R., Klaver, C. C., Hofman, A., and de Jong, P. T. 1995b. Epidemiology of age-related maculopathy.

Epidemiol Rev 17:347-360.

Weller, R. O., and Nicoll, J. A. 2003. Cerebral amyloid angiopathy: pathogenesis and effects on the ageing and Alzheimer brain. Neurol Res 25:611-616.

Yannuzzi, L. A., Negrao, S., Iida, T., Carvalho, C., Rodriguez-Coleman, H., Slakter, J., Freund, K. B., Sorenson, J., Orlock, D., and Borodoker, N. 2001. Retinal angiomatous proliferation in age-related macular degeneration. Retina 21:416-434.