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

Accumulation of protein- and lipid-containing deposits external to the retinal pigment epithelium (RPE) is common in the aging eye, and has long been viewed as the hallmark of age-related macular degeneration (AMD). The cause for the accumulation and retention of molecules in the sub-RPE space, however, remains an enigma. Here, we present fluorescence microscopy and X-ray diffraction evidence for the formation of small (0.5-20 μm in diameter), hollow, hydroxyapatite (HAP) spherules in Bruch's membrane in human eyes. These spherules are distinct in form, placement, and staining from the well-known calcification of the elastin layer of the aging Bruch's membrane. Secondary ion mass spectrometry (SIMS) imaging confirmed the presence of calcium phosphate in the spherules and identified cholesterol enrichment in their core. Using HAP-selective fluorescent dyes, we show that all types of sub-RPE deposits in the macula, as well as in the periphery, contain numerous HAP spherules. Immunohistochemical labeling for proteins characteristic of sub-RPE deposits, such as complement factor H, vitronectin, and amyloid beta, revealed that HAP spherules were coated with these proteins. HAP spherules were also found outside the sub-RPE deposits, ready to bind proteins at the RPE/choroid interface. Based on these results, we propose a novel mechanism for the growth, and possibly even the formation, of sub-RPE deposits, namely, that the deposit growth and formation begin with the deposition of insoluble HAP shells around naturally occurring, cholesterol-containing extracellular lipid droplets at the RPE/choroid interface; proteins and lipids then attach to these shells, initiating or supporting the growth of sub-RPE deposits.