Purpose: To determine the mechanism of reattachment of harvested human retinal pigment epithelium (RPE) to RPE-derived extracellular matrix and Bruch's membrane.
Methods: Confluent first-to third-passage human RPE were harvested from tissue culture and plated onto RPE-derived extracellular matrix or human Bruch's membrane exoplants denuded of cells by treatment with 0.02 N ammonium hydroxide. The authors measured RPE reattachment to uncoated surfaces or surfaces precoated with extracellular matrix proteins (fibronectin, laminin, vitronectin, or type IV collagen), antibodies to extracellular matrix-proteins, or the synthetic peptide RGDS (arginine-glycine-aspartate-serine). Some RPE were pretreated with anti-beta 1 integrin antibodies before plating onto either substrate.
Results: Coating the surface of either RPE-derived extracellular matrix or Bruch's membrane with fibronectin, laminin, vitronectin, or type IV collagen increased the RPE attachment rate. Exposing RPE to anti-beta 1 integrin antibodies or RGDS or precoating the surface with antibodies to fibronectin, laminin, vitronectin, or type IV collagen decreased the RPE attachment rate to both surfaces. The RPE attachment rate to Bruch's membrane was lower when the exoplants were harvested from the macula of older (age, 70 to 90 years) versus younger (age, 30 to 40 years) persons (52.4 +/- 3.6% versus 64.3 +/- 3.5%, respectively; P < 0.05).
Conclusions: The attachment of cultured human RPE cells to human Bruch's membrane or to RPE-derived extracellular matrix is mediated by an interaction between the beta 1-subunit of integrin on the RPE surface and ligands in the extracellular matrix that include laminin, fibronectin, vitronectin, and type IV collagen. The lower rate of RPE reattachment to the macula from older human cadaveric eyes may have implications for studies aimed at RPE transplantation in elderly persons.