Regulation of Corneal Endothelial Cell Behavior through Manipulation of Cellular Microenvironment

Abstract

Purpose: Human diseases of the corneal endothelium are often accompanied with, or caused by, changes in the molecular composition of its extracellular matrix (ECM). We have previously reported the importance of laminin β2 and γ3 chains in maintaining endothelial cell monolayer formation and thickness of Descemet’s Membrane (DM) in mouse cornea. Our aim is to understand the specific substrates that yield optimal endothelial cell monolayer formation and physiological behavior when seeded onto engineered scaffolds.

Methods: The distribution of laminin chains was studied via immunohistochemistry (IHC) in sections of mouse and bovine cornea. Additionally, commercially available and primary corneal endothelial cells from various species were cultured and seeded onto a variety of laminin heterotrimers including: Ln521, Ln411, and Ln332. These molecules were used to coat glass coverslips and specially fabricated scaffolds of differing composition. Cells were allowed to grow for a series of specified times and were then assayed for tight junction formation via anti-ZO-1 and β-catenin IHC. Data were compared with cells grown on uncoated, poly-D-Lysine-coated, and Matrigel-coated coverslips.

Results: Out of the laminin chains tested, α3, β3, γ2, were confirmed in bovine DM, while β2 and γ3 were found in the limbus and peripheral regions of mouse corneas. Next, when we seeded commercially available bovine corneal endothelial cells (BCECs) onto uncoated or coated glass coverslips, we observed that adherens and tight junctions are formed no sooner than culture day 17 (D17), as shown by β-catenin and ZO-1 immunoreactivity (IR), respectively. In a parallel study, these cells were also seeded onto specially nanofabricated scaffolds comprised of SU8, and compared to commercially available polyester membranes. Adherens and tight-junction formation was observed on Ln332-coated SU8 after 20 days, compared to 8 days when seeded on laminin-coated polyester.

Conclusions: These data demonstrate that specific laminin chains, when coupled to various surfaces of differing physical properties, can be used to regulate endothelial cell differentiation. A full understanding of this aspect of endothelial behavior is critical for the development of fabricated biomimetic scaffolds.

Keywords: 481 cornea: endothelium • 519 extracellular matrix • 607 nanotechnology