A Comparison of Gene and Protein Expression in Primary Human Trabecular Meshwork Cells Cultured With Human Aqueous Humor or Fetal Bovine Serum

Abstract

Abstract: : Purpose:Proteins in aqueous humor are responsible for maintaining trabecular meshwork (TM) cells in a normal homeostatic environment. Changes in the types and amounts of proteins in aqueous humor may influence the genetic program of these cells altering their function. We have examined primary human TM cell cultures following incubation with human aqueous humor or fetal bovine serum (FBS; standard culture medium) to determine whether differences in gene and protein expression exist. Methods: Confluent primary human TM cells were incubated with DMEM containing either 50% human aqueous humor or 10% FBS. Following a 5 day incubation, total RNA was isolated from two independent TM cell lines, processed into cRNA, and used to probe Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays (n=2 for each media condition). Cell lysates at day 5 were also prepared for 2–dimensional gel electrophoresis. Array data was analyzed using Genespring and Ingenuity Pathway Analysis Programs. Protein expression in 2–dimensional gels was compared using PDQuest software. Cell counts were also performed at various timepoints over a 21 day period. Results: Primary human TM cells incubated in 50% human aqueous humor showed significant gene and protein expression changes when compared to cells incubated in 10% FBS. Approximately 10% of the 15,000 expressed genes showed greater than 2–fold changes. In particular, genes associated with cell growth and proliferation, cell signaling, cell movement and cell cycling were altered. Confluent TM cells incubated in 50% human aqueous humor showed an 11% increase in cell numbers over 21 days similar to expected findings in vivo where TM cells undergo little or no cell proliferation. In contrast, cells incubated with 10% FBS increased by 120% over the same period. Conclusions: Primary human TM cells cultured in aqueous humor produce a molecular profile that differs significantly from cells placed in FBS (standard culture medium). As methods become more sophisticated and are able to identify regulation of gene networks, it will be important for cultures such as primary TM cells to display profiles similar to the in vivo state. Therefore, the need to incubate primary TM cells in a medium that contains the types and proportions of proteins present in aqueous humor may be necessary to maintain a homeostatic environment.

Keywords: trabecular meshwork • gene microarray • proteomics