July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Amelotin, a promoter of hydroxyapatite mineralization, is induced in serum-deprived RPE cells and colocalizes with calcium deposits in AMD eyes.
Author Affiliations & Notes
  • Dinusha Rajapakse
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Katherine Peterson
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Sanghamitra Mishra
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
  • Graeme Wistow
    National Eye Institute, National Institutes of Health, Bethesda, Maryland, United States
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2438. doi:
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      Dinusha Rajapakse, Katherine Peterson, Sanghamitra Mishra, Graeme Wistow; Amelotin, a promoter of hydroxyapatite mineralization, is induced in serum-deprived RPE cells and colocalizes with calcium deposits in AMD eyes.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2438.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Hydroxyapatite spherules containing lipid and protein accumulation between the basal lamina of the Retinal pigment epithelium (RPE) and Bruch’s membrane are common in the aging eye and are viewed as a hallmark of Age-relate Macular Degeneration (AMD). However, the mechanism of hydroxyapatite mineralization in the eye is yet unclear.

Methods : A three-dimensional RPE cell culture model was developed using serum deprivation for days 0-9 compared to control RPE cells cultured in 10% serum. RPE monolayers were fixed and sectioned for confocal microscopy.
RNA samples of the serum deprived RPE from days 0-9 (N=3) were sequenced by the NIH Intramural Sequencing Center using the Illumina HiSeq platform. Analysis of the sequence to identify differentially expressed genes was performed by the BIMAS group at the NIH Center for Information Technology using the DESeq analysis program.
Normal and AMD-affected eyes (N=4) from NDRI were fixed and sectioned through the macula for confocal microscopy.

Results : An enamel protein, Amelotin (AMTN), exhibits a 60-fold increase in expression at Day 9 of serum depravation compared to Day 0 in 10% serum (P< 0.05). Western blot shows Amelotin protein expression at Day 9. Immunocytochemistry of RPE sections confirmed Amelotin expression in serum deprived RPE at Day 9 and hydroxyapatite deposition in the basal regions of RPE monolayer. While normal donor eye showed no immunofluorescence labelling for amelotin, AMD donor eyes stained positive for amelotin and hydroxyapatite/calcium deposits in hard and soft drusen.

Conclusions : Our results have identified the enamel matrix protein, Amelotin which has been shown to promote hydroxyapatite mineralization in dental enamel to be expressed in RPE cells as well as in drusen in AMD eyes. These finding are consistent with our hypothesis that Amelotin has a potential role in hydroxyapatite formation in the aging eye. Further investigations are necessary to completely understand the mechanisms leading to hydroxyapatite mineralization in the eye.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Patterns of localization of amelotin and hydroxyapatite spherules in a H/H AMD human donor eye with large drusen.

Patterns of localization of amelotin and hydroxyapatite spherules in a H/H AMD human donor eye with large drusen.

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