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R. Hamilton, L. Leach, A. Foss; Characterisation of a Human in vitro Tri–Layer to Mimic the Outer Blood – Retinal Barrier . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3044.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Reductionist approaches into processes underlying age related macular degeneration (ARMD) have been hampered by the lack of optimal in vitro human models. We therefore investigated whether co–culture of retinal pigment epithelium (RPE) and human umbilical vein endothelial cells (HUVEC) on either side of human amnion would result in a tri–layer which mimics the in vivo RPE – Bruch’s membrane – choriocapillaris in junctional integrity, permeability and morphology. Methods: Dynamics of tight (TJ) and adherens (AJ) junctional molecules were analysed by immunocytochemistry and imaging. Tri–layer permeability was tested by tracer leakage studies using 4kDa fluorescein–conjugated dextran under flow. Results: Tri–layers of HUVEC and RPE cells on amnion remained viable for 9 days with no discernible morphological changes. The TJ molecule, zonula occludens–1 and the AJ molecule, vascular endothelial cadherin were localised to cell–cell contacts of RPE and HUVEC respectively from 24 hours, and remained unaltered thereafter. Initially the trans–membrane TJ marker, occludin was cytoplasmic but localised to cell – cell contacts of both cell types after 48hrs and was fully junctional at 72 hrs; this was not achieved in monocultures. At 72 hrs, HUVEC or RPE cells on amnion respectively were leaky to 4kDa dextran. However the tri–layer, concomitant with increased junctional occludin, severely restricted/occluded the tracer, confirming the presence of functional tight junctions. Ultrastructural studies showed the presence of epithelial TJs and a fenestrated endothelial layer. Conclusions: This study demonstrates the importance of cross talk between epithelial and endothelial layers in achieving phenotypes which resemble that in vivo. Junctional occludin appears to be pivotal in determining restrictivity of the barrier. The tri–layer provides a model which can be used to study induced junctional disruption and vascular remodelling: features of ARMD.
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