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Sabrina Cahyadi, Bhavika J. Patel, Meihua Ju, Alexia Phedonos, Anishka Wilwaraarachchi, Imre Lengyel; Altered Zinc Balance can Directly Affect Fenestrae Formation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):522.
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© ARVO (1962-2015); The Authors (2016-present)
Altered zinc balance had been associated with age related macular degeneration (AMD). The purpose of this study was to examine whether the alteration in zinc balance can directly affect the choroidal microvasculature through modulating fenestrae formation.
Fenestrae formation was studied in a murine endothelioma cell line (bEND5) that has been shown to produce fenestrae when treated with 1.5 uM Latrunculin A (LA) for 3 h (Ioannidou et al 2006) a treatment that were used as positive control in our experiments. Cells were seeded onto 1% gelatine coated cover slips and maintained in high-glucose DMEM supplemented with 10% FBS and antibiotics. Zinc treatment started on the second day and lasted for up to 20 hours. Rhodamine phalloidin staining was used to visualize F-actin rearrangement. Fenestrae formation was observed by monitoring the redistribution of diaphragm protein PV1 immunoreactivity using a Zeiss LSM700 confocal microscope and by whole-mount transmission electron microscopy.
Cells incubated with LA showed the characteristic pattern of changes in bEND5 cells. These include F-actin depolimerisation, PV1 rearrangement into the so called "sieve plates" and typical pore formation on TEM. When cells were exposed to up to 1 uM exchangeable extracellular zinc alone there was only a minor F-actin rearrangement, but clear signs of sieve-plate formation. TEM confirmed the presence of fenestrae in areas of sieve-plates.
We showed here for the first time that <1 uM extracellular exchangeable zinc can induce the formation of fenestrae in vitro. This might be relevant for the formation and/or maintenance of fenestrated choroidal micro capillaries in vivo. Once zinc balance is affected fenestrae formation and consequently appropriate clearance will be impaired, leading to sub-RPE deposits formation and AMD.
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