Purpose: Uncultured aged retinal pigment epithelium (RPE) does not resurface aged Bruch's membrane after 24 hours in organ culture. These experiments assess whether culturing alters RPE integrin expression and resurfacing of Bruch's membrane.
Methods: RNA was isolated from uncultured and cultured RPE of aged adult donor and fetal eyes. Integrin subunit messenger RNA (mRNA) expression was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and semiquantitative analysis of the amplified products. Cell surface integrin expression was assessed using flow cytometry. Passaged cultured fetal RPE and primary cultured aged RPE were seeded onto Bruch's membrane, and resurfacing was assessed with scanning electron microscopy.
Results: Uncultured fetal RPE had low levels of alpha3 and beta5 mRNA compared to passaged cultured fetal RPE. Uncultured aged RPE had decreased alpha1-5 mRNA compared to primary cultured aged RPE. Cultured aged RPE had decreased beta4 and beta5 mRNA compared to passaged cultured fetal RPE. Flow cytometry confirmed the expression of alpha1-5, alphav, and beta1 protein on cultured fetal RPE and alpha1-3 and beta1 protein on cultured aged RPE. Twenty-four hours after seeding, cultured fetal and aged RPE resurfaced 99% +/- 1.3% and 76% +/- 22%, respectively, of aged submacular Bruch's membrane specimens from which native RPE had been debrided, exposing the native RPE basement membrane. Cultured fetal and aged RPE resurfaced 97% +/- 3.1% and 39% +/- 35%, respectively, of specimens in which the inner collagenous layer was exposed.
Conclusions: Uncultured aged RPE has low amounts of integrin subunits that form receptors for laminin, fibronectin, and collagens. Culturing up-regulates integrins and promotes more efficient aged RPE attachment to and survival on aged Bruch's membrane.