Cell Culture Model That Mimics Drusen Formation and Triggers Complement Activation Associated with Age-Related Macular Degeneration
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Cell culture model that mimics drusen formation and triggers complement activation associated with age-related macular degeneration Lincoln V. Johnsona,1, David L. Foresta, Christopher D. Bannaa, Carolyn M. Radekea, Michelle A. Maloneya, Jane Hub, Christine N. Spencera, Aimee M. Walkera, Marlene S. Tsiea, Dean Bokc, Monte J. Radekea, and Don H. Andersona aCenter for the Study of Macular Degeneration, Neuroscience Research Institute, University of California, Santa Barbara, CA 93106; and bDepartment of Ophthalmology and cDepartment of Neurobiology, Brain Research Institute, Jules Stein Eye Institute, The David Geffen School of Medicine, University of California, Los Angeles, CA 90095 Edited* by Jeremy Nathans, The Johns Hopkins University, Baltimore, MD, and approved September 9, 2011 (received for review June 15, 2011) We introduce a human retinal pigmented epithelial (RPE) cell-culture genesis, as well as the relevant molecular interactions leading to model that mimics several key aspects of early stage age-related drusen deposition, have not yet been fully elucidated. macular degeneration (AMD). These include accumulation of sub-RPE During the past decade, compelling evidence has emerged deposits that contain molecular constituents of human drusen, and implicating the immune system—and the complement system in activation of complement leading to formation of deposit-associated particular—in drusen biogenesis and AMD (15, 20, 21). A terminal complement complexes. Abundant sub-RPE deposits that number of the proteins detected in drusen are either comple- are rich in apolipoprotein E (APOE), a prominent drusen constituent, ment components or related molecules. Importantly, variations are formed by RPE cells grown on porous supports. Exposure to in several complement-related genes have been shown to be human serum results in selective, deposit-associated accumulation of highly significant risk factors for the development of AMD (20, additional known drusen components, including vitronectin, clus- 21). Taken together, these findings are consistent with the gen- terin, and serum amyloid P, thus suggesting that specificprotein– eral conclusion that chronic local inflammation at the RPE/ protein interactions contribute to the accretion of plasma proteins Bruch’s membrane interface contributes to drusen formation during drusen formation. Serum exposure also leads to complement and to AMD pathogenesis (12, 14, 22, 23). activation, as evidenced by the generation of C5b-9 immunoreactive Despite these significant advances, the identity of the mole- terminal complement complexes in association with APOE-contain- cules responsible for triggering activation of the complement ing deposits. Ultrastructural analyses reveal two morphologically cascade, as well as the downstream molecular interactions that distinct forms of deposits: One consisting of membrane-bounded promote AMD pathology, remain elusive. This is due, in part, to multivescicular material, and the other of nonmembrane-bounded the dearth of animal and cell-culture models that reproduce the particle conglomerates. Collectively, these results suggest that dru- most salient pathologic features of AMD under controlled ex- sen formation involves the accumulation of sub-RPE material rich in perimental conditions. We introduce here an RPE cell culture APOE, a prominent biosynthetic product of the RPE, which interacts model that mimics numerous aspects of AMD pathology ob- with a select group of drusen-associated plasma proteins. Activation served in humans, including accumulation of sub-RPE deposits of the complement cascade appears to be mediated via the classical containing known constituents of drusen, activation of the com- pathway by the binding of C1q to ligands in APOE-rich deposits, plement system, and deposition of terminal complement com- triggering direct activation of complement by C1q, deposition of plexes. This system provides a unique experimental platform that terminal complement complexes and inflammatory sequelae. This model system will facilitate the analysis of molecular and cellular will facilitate dissection of the cellular and molecular events that lead to drusen formation and contribute to AMD pathogenesis. aspects of AMD pathogenesis, and the testing of new therapeutic CELL BIOLOGY agents for its treatment. Results Examination of differentiated cultures of primary human RPE ge-related macular degeneration (AMD) is characterized in cells grown on porous supports led to the identification of a pop- Aits early stages by the presence of extracellular deposits, ulation of globular extracellular deposits that accumulate within known as drusen, that accumulate between the basal surface of the pores of the support material. Initially, we identified these sub- the retinal pigmented epithelium (RPE) and Bruch’s membrane, RPE deposits based on their immunoreactivity for apolipoprotein an extracellular matrix complex that separates the neural retina E (APOE) (Fig. 1). Antibodies to several other apolipoproteins, from the capillary network in the choroid. Early electron mi- including apolipoproteins A and B, did not show similar immu- croscopic studies suggested that drusen formation may be noreactivity. APOE is a circulating lipid transport protein, a a consequence of degeneration of the RPE (1–3), initiated by ubiquitous component of human ocular drusen (24–26), and an membranous debris shed from its basal surface (4, 5). These abundant biosynthetic product of RPE cells (16, 24). The APOE early morphological observations have since been confirmed by deposits are typically spheroid, sometimes tubular in shape, a number of more recent studies (6–13). Contemporary investigations of the molecular composition of drusen have provided additional insights into their biogenesis. Author contributions: L.V.J., D.L.F., C.D.B., C.M.R., M.A.M., J.H., D.B., M.J.R., and D.H.A. Immunohistochemical and proteomic studies show that drusen designed research; L.V.J., D.L.F., C.D.B., C.M.R., M.A.M., J.H., C.N.S., A.M.W., and M.S.T. performed research; L.V.J., D.L.F., C.D.B., C.M.R., M.A.M., J.H., C.N.S., A.M.W., M.S.T., D.B., contain a variety of protein and lipid components (14, 15). Among M.J.R., and D.H.A. analyzed data; and L.V.J. and D.H.A. wrote the paper. these are several plasma proteins, the presence of which implies The authors declare no conflict of interest. a systemic contribution to their genesis. Although the primary *This Direct Submission article had a prearranged editor. biosynthetic source for most of these circulating molecules is the Freely available online through the PNAS open access option. liver, a number of them are also known to be synthesized locally by 1To whom correspondence should be addressed. E-mail: [email protected]. – RPE cells (15 19). The respective contributions of RPE-derived This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and plasma-derived molecules to the process of drusen bio- 1073/pnas.1109703108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1109703108 PNAS | November 8, 2011 | vol. 108 | no. 45 | 18277–18282 Downloaded by guest on September 24, 2021 Fig. 1. APOE-containing basal deposits are elaborated by human RPE cells in vitro. Confocal immunofluorescence images of sectioned (A and B) and flat- mounted (C) RPE cell cultures on porous supports. Spherical to amorphous deposits that are immunoreactive for APOE (green, arrows) accumulate within the support matrix. These deposits are nonuniformly distributed with areas of high deposit density (arrows in A and C) interspersed among areas with few to no detectable deposits (asterisk in A). Immunoreactivity for GPNMB (red in A), an abundant RPE membrane protein, is localized to RPE cell surfaces and to par- ticulate sub-RPE material (arrowheads in A) that is distinct from the APOE deposits. Blue, nuclear (“N” in B) DAPI fluorescence in A and B. (Scale bars, 10 μminA and C;5μminB.) ranging from 0.5 to 3 μm in diameter. They accumulate within the assayed immunocytochemically for C5b-9 show no evidence of porous culture matrix and are most concentrated within 20 μmofthe immunoreactivity (Fig. S1), indicating that neither components of basal surface of the RPE. The deposits are nonuniformly distributed the medium nor the support matrix are responsible for comple- along the substratum, with areas of higher density interspersed with ment activation and deposition of C5b-9 complexes. areas where the deposits are relatively sparse (Fig. 1). This sporadic By electron microscopy, two morphologically distinct deposit distribution of sub-RPE material is consistent with the irregular subtypes are identified: nonmembrane-bounded conglomerates pattern of drusen development in the eye. The accumulation of of small osmophilic particles, and membrane-bounded, multi- APOE from bovine serum that is a constituent of the RPE cell- vesicular structures (Fig. 3). These two subtypes are sometimes culture medium does not appear to contribute to the APOE im- segregated, and sometimes comingled within deposits. munoreactivity because cell culture inserts without cells incubated in We assessed the relative contributions of the different com- bovine serum-containing medium and assayed immunocytochemi- plement activation pathways to deposit-associated formation of cally for APOE show no evidence of immunoreactivity (Fig. S1). C5b-9 terminal complement complexes by exposing cultures to The APOE-containing deposits also show comparatively low human sera depleted of specific components required