Differential Pattern of Junctional Complex Lipid Raft Proteins in Human Conventional Outflow

Abstract

 

Purpose:

 

The trabecular meshwork (TM) is a specialized tissue forming a biologic filter for aqueous humor. The two dominant cell types in the TM are Schlemm’s canal (SC) endothelial cells on the inner wall of the collecting channels and TM cells that cover the TM beams. SC endothelial cells differ morphologically and functionally from TM endothelial cells. Exactly how these cells, their junctional complexes or their extracellular matrices, regulate aqueous outflow barrier resistance is poorly understood. The purpose of this study was to compare junctional complex lipid raft proteins of SC cells to that of TM cells.

 

Methods:

 

Human SC and TM cells were grown to confluency in T-25 flasks. After two weeks at confluence, cells were lysed in ice cold lysis buffer containing 1% Triton X-100. Caveolin enriched lipid rafts were isolated using an OptiPrep density gradient (Sigma D1556). The preparation was centrifuged at 200,000 x g for 18 hrs; nine 1.0 ml fractions were pipetted from the top (lightest) to bottom (heaviest). Each fraction was analyzed for protein content. Equal amounts of proteins were resolved by SDS polyacrylamide gel electrophoresis and immunoblotted with a panel of junctional complex antibodies. Densitometry was performed.

 

Results:

 

Profile of junctional complexes in enriched lipid rafts.  

 

Conclusions:

 

This is the first identification and comparison of the junctional complexes in SC and TM. Lipid raft proteins in SC cells are distinct from TM cells. SC cells express more annexin 2 than TM cells. Both have considerable amounts of occludin, zona occludens and other constituents of the lipid rafts. These differential expression patterns likely correspond to differences in function of TM and SC endothelial cells in modulating aqueous outflow resistance.

 

Keywords: trabecular meshwork • cell adhesions/cell junctions • extracellular matrix