Cell culture model that mimics drusen formation and triggers complement activation associated with age-related macular degeneration

Abstract

We introduce a human retinal pigmented epithelial (RPE) cell-culture model that mimics several key aspects of early stage age-related macular degeneration (AMD). These include accumulation of sub-RPE deposits that contain molecular constituents of human drusen, and activation of complement leading to formation of deposit-associated terminal complement complexes. Abundant sub-RPE deposits that are rich in apolipoprotein E (APOE), a prominent drusen constituent, are formed by RPE cells grown on porous supports. Exposure to human serum results in selective, deposit-associated accumulation of additional known drusen components, including vitronectin, clusterin, and serum amyloid P, thus suggesting that specific proteinprotein interactions contribute to the accretion of plasma proteins during drusen formation. Serum exposure also leads to complement activation, as evidenced by the generation of C5b-9 immunoreactive terminal complement complexes in association with APOE-containing deposits. Ultrastructural analyses reveal two morphologically distinct forms of deposits: One consisting of membrane-bounded multivescicular material, and the other of nonmembrane-bounded particle conglomerates. Collectively, these results suggest that drusen formation involves the accumulation of sub-RPE material rich in APOE, a prominent biosynthetic product of the RPE, which interacts with a select group of drusen-associated plasma proteins. Activation of the complement cascade appears to be mediated via the classical pathway by the binding of C1q to ligands in APOE-rich deposits, triggering direct activation of complement by C1q, deposition of terminal complement complexes and inflammatory sequelae. This model system will facilitate the analysis of molecular and cellular aspects of AMD pathogenesis, and the testing of new therapeutic agents for its treatment.A ge-related macular degeneration (AMD) is characterized in its early stages by the presence of extracellular deposits, known as drusen, that accumulate between the basal surface of the retinal pigmented epithelium (RPE) and Bruch's membrane, an extracellular matrix complex that separates the neural retina from the capillary network in the choroid. Early electron microscopic studies suggested that drusen formation may be a consequence of degeneration of the RPE (1-3), initiated by membranous debris shed from its basal surface (4, 5). These early morphological observations have since been confirmed by a number of more recent studies (6-13).Contemporary investigations of the molecular composition of drusen have provided additional insights into their biogenesis. Immunohistochemical and proteomic studies show that drusen contain a variety of protein and lipid components (14, 15). Among these are several plasma proteins, the presence of which implies a systemic contribution to their genesis. Although the primary biosynthetic source for most of these circulating molecules is the liver, a number of them are also known to be synthesized locally by RPE cells (15)(16)(17)(18)(19). The respect...

Smoke Exposure Causes Endoplasmic Reticulum Stress and Lipid Accumulation in Retinal Pigment Epithelium through Oxidative Stress and Complement Activation

“…Here we provide data that suggests that cholesterol uptake regulated via the transcription factors SREBF-1, and triggered by complement activation, might be involved in lipid dysregulation in the RPE. Interestingly, similar to the work reported here, Johnson and co-workers (72) showed that primary RPE cells grown in long term cultures accumulate sub-RPE deposits rich in apolipoprotein E. Taken together, whereas the data in the literature as well as our data do not provide clear evidence that the lipid deposits are derived from reprocessed serum lipids rather than from RPE plasma membrane, these cumulative data clearly support the hypothesis that photoreceptor phagocytosis is not required. We hope to determine the source of lipids in future studies.…”

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye

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Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype

“…However, using factor B-depleted serum, we did observe C1q-dependent deposition of C5b-9 within the culture insert filters below the hRPE cells (Fig. 4), as described previously (40). Deposition of C5b-9 within the nitrocellulose filters was seen in hRPE cultured cells of both CFH haplotypes regardless of the OS bisretinoid content (Fig.…”