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Richard B Thompson, Valentina Reffatto, Jacob Bundy, Jane Flinn, Antonio Lanzirotti, Emrys Jones, David McPhail, Savanjeet Ratu, Alan C Bird, Imre Lengyel; A novel mechanism for initiation of sub-RPE deposits. Invest. Ophthalmol. Vis. Sci. 2014;55(13):623.
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It remains unclear how sub-RPE deposits in the human eye start to form. Here we present evidence that hydroxyapatite (HAP), the highly insoluble mineral component of bone, is present in human sub-RPE deposits as small spherules, and HAP “seeds” the deposition of proteins and lipids.
Donor eyes were obtained from the Eye Bank at Moorfields Eye Hospital less than 24 hours post mortem. Areas with sub-RPE deposits were dissected. Following the removal of the neuronal retina and the retinal pigment epithelium, the remaining choroidal microcapillaries/Bruch’s membrane/sub-RPE deposit complex was flat mounted. These were analyzed by microprobe synchrotron X-ray fluorescence (SXRF) and x-ray diffraction (XRD) at the X27A beam line at the National Synchrotron Light Source at Brookhaven National Laboratory. In addition, flat-mounted tissues were stained with fluorescent dyes selective for hydroxyapatite or immunostained and imaged by confocal fluorescence microscopy. The deposits were also analyzed by time-of-flight secondary ion mass spectrometry (TOF-SIMS) following focused ion beam milling of the specimen.
The XRD data unambiguously identified hydroxyapatite in sub-RPE deposits. The use of two in situ fluorescent stains for HAP showed spherular staining in all forms of sub-RPE deposit and indicated that HAP spherules are present on the BM even in areas where there are no visible drusen. The average size of the HAP spherules is ~3 um. Usually numerous spherules are distributed throughout individual sub-RPE deposits, including some that are coated with previously identified drusen proteins, including complement factor H, vitronectin, and amyloid beta. TOF-SIMS imaging confirmed the presence of Ca and PO4 in the spherules, as well as protein on their surfaces, and cholesterol in their centers.
The unexpected presence of hydroxyapatite spherules indicates that proteins might bind to HAP spherules in vivo at the RPE/choroid interface in the manner of HAP as a chromatographic stationary phase. Understanding these interactions may provide a means to forestall or reverse deposit formation well before irreversible sight loss occurs.
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