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K. G. Howell, A. A. Leontovich, S. Raghavakaimal, D. H. Johnson, M. P. Fautsch; Elevated Levels of TGFß2 Cause Unique Gene Expression Changes in Trabecular Meshwork Cells Incubated in Human Aqueous Humor. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2060.
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TGFß2 is a multifunctional protein having stimulatory or inhibitory activity on many cellular mechanisms. The action of TGFß2 is dependent on the presence or absence of other cell signaling molecules present in the extracellular matrix or surrounding milieu. In primary open angle glaucoma, TGFß2 levels in aqueous humor are elevated in approximately 50% of patients. In this study, we determined the effect of elevated levels of TGFß2 on trabecular meshwork (TM) gene expression following incubation in human aqueous humor or fetal bovine serum.
Three independent confluent primary human TM cell lines were incubated with DMEM (Dulbecco’s Modified Eagle’s Media) containing either 50% human aqueous humor or 10% fetal bovine serum in the presence or absence of elevated levels of TGFß2 (3 ng/ml). Following five day incubations, total RNA was isolated from each cell line and condition. Total RNA (100 ng) was amplified twice and processed into cRNA. cRNA from each cell line and condition was used to probe Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays (n=3 for each media condition). Array data was analyzed using Genespring and Genego Analysis Programs (Metacore). Only sequences with a 2-fold change in expression and a p < 0.05 were used in the analysis.
Primary human TM cells incubated in 50% aqueous humor or 10% fetal bovine serum containing elevated levels of TGFß2 showed significant gene expression changes when compared to cells incubated in 50% aqueous humor or fetal bovine serum alone. Only 98 sequences were differentially expressed in both aqueous humor and fetal bovine serum in the presence of elevated levels of TGFß2 (i.e. versican, etc). In aqueous humor, TGFß2 altered expression of 215 unique sequences. Fibronectin was induced by TGFß2 in aqueous humor but no change was seen in fibronectin expression in TM cells incubated in 10% fetal bovine serum and TGFß2. Our studies in 50% human aqueous humor show the addition of elevated levels of TGFß2 affect cellular processes associated with cell adhesion, extracellular matrix formation, cell-cell and cell-matrix signaling.
Primary human TMcells cultured in human aqueous humor containing elevated levels of TGFß2 produce a unique molecular profile that differs from cells cultured in either aqueous humor or fetal bovine serum alone. The identification of this specific gene set may lead to new ideas as to the potential effect that elevated levels of TGFß2 have on TM cell homeostasis.
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