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S. Aisenbrey, J. Yee, M. Koch, W.H. Brunken, D.D. Hunter; RPE Cells Synthesize and Preferentially Adhere to Laminin 5, a Potential Stabilizing Component of Bruch’s Membrane . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5158.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Macular degenerations are characterized by alterations in the structure of Bruch’s membrane (BrM) leading to changes in adhesion properties, perhaps via changes in interactions with extracellular matrix (ECM) proteins. In vitro, RPE cells express ECM proteins including type IV collagens and laminins, and can form a matrix. We previously showed that RPE cells synthesize several laminin chains in vivo that could potentially form several laminin heterotrimers including laminin 5. Adhesion properties and cell characteristics of RPE cells are dependent on the substrate they are cultivated on. The aim of this study is to identify specific laminins expressed by RPE cells in vitro and test substrates for cell adhesion accordingly. Methods: Immunohistochemistry was performed on RPE cell lines (RPE–J, ARPE19) after 3 and 6 days in culture using monoclonal and polyclonal antibodies against known laminin chains. Primers against laminin chains were designed and RT–PCR performed on RNA extracts of these cell lines. Cell adhesion assays were performed using EHS collagen IV, EHS laminin 1, purified laminin 5, and heterologously expressed "short arm" fragments of laminin ß2, laminin γ2, and laminin γ3 as substrates; cell attachment was quantified after fixation. Results: The laminin chains α1, α3, α5, ß1, ß2, γ1, γ2 are secreted by RPE cell lines in vitro. Similarly, RT–PCR of RNA from cell lines revealed the presence of RNA for laminin α1, α5, ß1, ß2, ß3, γ1, γ2, γ3. Together, these data suggest that RPE cells can synthesize laminins including laminin 1 (α1ß1γ1) and laminin 5 (α3ß3γ2), and perhaps several others. Therefore, we determined whether RPE cells preferentially adhered to these potential adhesive components of BrM. Although RPE cells adhered to laminin 1 and collagen IV, they preferred laminin 5. Conclusions: Specific laminins are expressed in RPE cells in vitro. RPE cells prefer laminin 5 to other known adhesive substrates. Clustering of specific integrin isoforms and other focal adhesion components by different laminins has to be shown. Composition of matrix molecules is likely to play a critical role in diseases involving the Bruch’s membrane–RPE complex and would, therefore, be critical for therapeutical approaches including RPE substitution.
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