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V.K. Gullapalli, I.K. Sugino, M.A. Zarbin; Role of Alpha 1–3 Integrin Subunits in Retinal Pigment Epithelial Attachment . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4148.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To examine the role of α1–3 integrin subunits in RPE adhesion to extracellular matrix and Bruch’s membrane. These subunits are expressed in low amounts in freshly harvested aged adult RPE compared to αv and others. Methods: Single suspensions of aged (≥ 55 yrs) adult RPE (1–2x105 cells/ml) cultures were pre–incubated with or without function blocking antibodies against α1–3, (10µg/ml each) either singly or in combination. Controls were incubated with normal IgG. Cells were seeded onto laminin– (n=5) or collagen I– (n=5) coated 96–well plates in duplicate wells. Poly–lysine–coated and bovine serum albumin–coated wells served as positive and negative controls respectively. After a 6–hour incubation, the number of attached cells was measured by a colorimetric assay. Localization of integrin subunits in focal adhesions was examined by immunofluorescence studies using antibodies to integrins and focal adhesion kinase. Cells pre–incubated with anti–α1–3 (n=4) or anti–α1–6 (n=3) antibodies were seeded onto RPE basement membrane (RPEbm) of submacular Bruch’s membrane explants. Cells pre–incubated with normal IgG and anti–αv were seeded on explants prepared from the fellow eye. After 6–hours, the number of rounded cells and spread cells were counted by scanning electron microscopy. Results: On laminin, blocking α1 decreased cell adhesion significantly. Combinations of antibodies resulted in further decrease only if one of the antibodies was anti– α1. On collagen I, blocking α1, 2, or 3 subunits singly resulted in a slight decrease that was not statistically significant. Blocking two or more subunits resulted in significant decrease compared to controls. Alpha 1–3 integrin subunits were found to co–localize with focal adhesion protein at sites of focal adhesions on both laminin and collagen–I. On submacular RPEbm, blocking α1–3 integrin subunits resulted in 45.7±10.5 % of cells spreading with the rest either rounded or broken up. In controls (αv and normal IgG), 62.6±5 % of the cells were able to attach and spread. The difference was statistically significant (p=0.027). Blocking α1–6 subunits resulted in 23.2±4.6 % of cells spreading on RPEbm versus 62.5±10.8% cells spreading following incubation with IgG and αv. Conclusions: Alpha 1–3 subunits are involved in RPE attachment to laminin and collagen I as well as to RPEbm. Absence of these subunits in freshly harvested adult RPE may hinder successful attachment and survival on aged Bruch’s membrane. αv appears to play a minimal, if any, role in RPE attachment to Bruch’s membrane.
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