Abstract
Purpose :
Amelotin (AMTN) is a protein originally identified in enamel formation in teeth. We discovered that AMTN can be induced in stressed retinal pigment epithelial (RPE) and is involved in the formation of hydroxyapatite (HAP) deposits in Age-related Macular Degeneration (AMD). The mechanism of AMTN induction is unknown, but we hypothesized that it may be part of a general response to injury in RPE.
Methods :
For scratch assays, RPE cells were cultured in chamber slides to create a confluent monolayer at 37 °C. After straight line scratch with a p200 pipet tip cells were incubated for 24 hours at 37 °C. Amelotin expression was detected by immunocytochemistry and calcium flux was imaged with fluo4-AM and fura-red-AM. For laser injury of retina, 4-month-old mice were anesthetized with Ketamine (90-120 mg/kg) and Xylazine (8-10mg/kg) intraperitoneally. Eyes were dilated with 1% tropicamide and 0.5% phenylephrine eye drops. The animals were positioned in a slit lamp delivery system with an OcuLight GLx ophthalmic laser to rupture Bruch's membrane (50µm spot size, 0.1 sec duration, ±80 mW (low laser) and ±120 mW (high laser) of power). Production of a tiny vaporization bubble immediately post-treatment indicated rupture of Bruch's membrane. Retina tissue were collected at time points 1, 3, 5, 7 and 14 days post laser injury. Cryosections of lasered and contralateral control mouse retinas were imaged by confocal microscopy using anti-Amelotin antibody and IRDye ® 800CW BoneTag HAP dye.
Results :
Scratch assays showed that AMTN is induced in RPE cells 24 hours post scratch. Laser injury in mouse retina showed induction of AMTN in both the RPE and adjacent photoreceptors surrounding laser lesions. AMTN expression peaked at day 3 and decreased as the wounds healed by day 14. HAP positive staining was identified in a large lesion 5-days post laser injury.
Conclusions :
AMTN is induced in RPE and photoreceptor cells in response to damage to RPE and Bruch’s membrane. Our findings suggest AMTN is involved in response to retinal injury, perhaps with a role in wound healing.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.