Culture-induced increase in alpha integrin subunit expression in retinal pigment epithelium is important for improved resurfacing of aged human Bruch's membrane

Abstract

The purpose of this study was to examine the change in integrin expression in adult human retinal pigment epithelium (RPE) after culturing and to characterize the role of integrins in RPE adhesion to aged submacular human Bruch's membrane. Expression of α integrin subunits 1 through 6 in adult RPE cells, cultured or uncultured, was examined by reverse transcription/real-time polymerase chain reaction (PCR) and Western blotting. RPE was cultured on bovine corneal endothelial cell-secreted extracellular matrix (BCE-ECM). The role of α integrin subunits in RPE attachment was examined by immunofluorescent localization of these subunits at sites of focal adhesions in cultured adult RPE attached to laminin or collagen-I-coated culture dishes. Additionally, the effect of function-blocking antibodies to α integrin subunits on RPE attachment to laminin, collagen I, and aged submacular human Bruch's membrane was determined. Cultured adult RPE had increased expression of α1–5 integrin subunits by PCR compared to uncultured RPE. Western blots showed that α2, 3, and 5 subunit levels were low or absent in uncultured adult RPE. Cultured adult RPE had a substantially higher expression of these integrins. Alpha 1–3 subunits co-localized with phosphorylated focal adhesion kinase (FAK) at focal adhesions in RPE cells spread on laminin. Only α2 and α3 co-localized with phosphorylated FAK in focal adhesions of RPE on collagen I. Using function blocking antibodies, blocking α1 subunit singly or in combination with α2 and/or α3 significantly decreased RPE adhesion to laminin. Blocking α1 and α2 or blocking α1, α2, and α3 subunits significantly decreased RPE adhesion to collagen I. Compared to controls, significantly fewer RPE cells were able to spread on aged submacular human Bruch's membrane when α1–6 integrin subunits were blocked. These results indicate that alpha 1–5 subunits that are upregulated by culturing on BCE-ECM are necessary for RPE attachment to aged submacular human Bruch's membrane. Relative lack of these integrin subunits in uncultured adult RPE may be responsible for poor resurfacing of aged submacular human Bruch's membrane by these cells.