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VK Gullapalli, IK Sugino, RB Birge, MA Zarbin; Integrin Expression In Human Retinal Pigment Epithelium (RPE): A Comparative, Semi-Quantitative RT-PCR Study Between Fetal And Adult Cells In Cultured And In Situ States . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3436.
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Purpose: Proper adhesion to Bruch's membrane (BrM) is one of the significant factors for successful transplantation of RPE in age-related macular degeneration. Cultured human fetal RPE attach to BrM more efficiently than freshly-isolated aged adult cells. The purpose of this study was to compare the expression of cell-substrate receptors, viz., integrins, between fetal and adult RPE. Methods: For studying expression in situ, RNA was extracted from RPE that was isolated into Trizol from fetal (n=4) and adult (n=5) donor eyes. For cultures, sheets of RPE obtained by collagenase type IV (adult eye: 0.4mg/ml; fetal eye: 0.8 mg/ml) digestion of posterior segment for approximately 90 minutes were triturated and cultured on bovine corneal endothelial extracellular matrix-coated (BCE-ECM) dishes. RNA was extracted from primary cultures of adult RPE (n=4) and second to fourth passage fetal cultures (n=4). Digestion with DNAse I was followed by reverse transcription-polymerase chain reaction (RT-PCR) with gene-specific primers using Superscript one-step RT-PCR kit (Gibco). Integrin subunits studied included α-1 to -6, αv, ß1, and ß-4 to ß-6. Negative controls included reaction mixtures with no RNA or no primers. PCR products were separated on 1.5% agarose gel, stained, and scanned using a fluorimager. Intensities (relative to actin) were compared. Results: Expression of α-1 to -5 was low to moderate in adult in situ while only α3 was low in in situ fetal. Alpha -1 to -5 were significantly upregulated in adult RPE upon culturing on BCE-ECM plates. Similar upregulation of α3 was seen in fetal cultures while the expression of the rest of the integrin subunits was not significantly different from in situ fetal RPE. Expression of ß4 was high in fetal cultures but low in other three categories. ß6 was absent in in situ adult. Conclusion: Since α1-5 are part of integrin receptors for laminin, collagen and fibronectin, freshly-isolated aged-adult RPE do not have the requisite receptors in sufficient amounts to attach and survive on Bruch's membrane. Cultured adult RPE or fetal RPE may be more suitable for transplantation.
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