Role of TGF-β2 in open-angle glaucoma becoming clear

October 1, 2007

Transforming growth factor beta (TGF-β2) increases outflow resistance in human anterior segments. Recent analysis has identified several candidate molecules and pathways by which elevated levels of TGF-b2 may trigger changes in outflow resistance.

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Rochester, MN-Transforming growth factor beta (TGF-β) is known to increase outflow resistance, and recent analysis has confirmed that TGF-β2 alters gene expression in molecules associated with several pathways. With further analysis, it may be possible to identify early molecular events associated with TGF-β2 and increased outflow resistance, according to Michael P. Fautsch, PhD, associate professor of ophthalmology, Mayo Clinic College of Medicine, Rochester, MN.

The TGF-β family has more than 40 members, and the three most commonly found in mammals are TGF-β1, TGF-β2, and TGF-β3. These molecules are involved in many cellular processes and may stimulate or inhibit certain processes, depending on the environment, Dr. Fautsch said. At least five studies have reported elevated levels of TGF-β2 in the aqueous humor of patients with primary open-angle glaucoma. In addition, two experimental studies have shown that TGF-β2 increases outflow resistance in perfusion organ culture.

Building on these findings, Dr. Fautsch set out to determine whether elevated levels of TGF-β2 changed IOP in the presence of porcine aqueous humor (50%) and to learn which genes and associated pathways in the trabecular meshwork are changed by TGF-β2.

Little change was seen in IOP in the control eyes over time, Dr. Fautsch reported. In eyes perfused with TGF-β2, IOP rose significantly early on; this increase was maintained throughout the length of the experiment.

At the 9-hour time-point, mean change in outflow resistance was 44% in the TGF-β2 eyes and 8% in the control eyes (p = 0.001, n = 11). At 96 hours, the changes were 43% and –15%, respectively (p = 0.013, n = 6).

The investigators then began to identify the genes and associated pathways involved in the changes observed in the TGF-β2 eyes. They used two pairs of eyes each that had undergone 9 and 96 hours of incubation, dissected the trabecular meshwork, isolated the total RNA, and used a microarray (U133 Plus 2.0, Affymetrix) that contains more than 54,000 sequences, to identify differential gene expression. The microarray data were validated with quantitative real-time polymerase chain reaction.

A variety of molecular changes were found. For example, expression of chemokine, ligand 5, had a moderate increase (2.6) in the 9-hour array but had increased by 40.7 at 96 hours. In contrast, MMP12 increased by 12.5 at 9 hours but had decreased to 1.0 by 96 hours.

In all, 431 unique sequences had a mean change of 1.5-fold by 9 hours, and 848 unique sequences changed by 96 hours. Only 88 sequences were either upregulated or downregulated at both 9 and 96 hours, Dr. Fautsch said.

Using a software suite for functional analysis of experimental data (MetaCore, GeneGo Inc.), the investigators obtained a report on the statistically significant pathways in their data. At 9 hours, chemokines exhibited a statistically significant change, particularly those in the CXCL family, which are involved in the inflammatory process.

"Interestingly enough, these molecules are mostly downregulated, suggesting that we are actually reducing inflammatory processes," Dr. Fautsch said. "The interleukins were also downregulated, so it appears that TGF-β2 does have an immunosuppressive effect and that we are decreasing the levels of inflammation."

Changes also were seen in JAM2 (junctional adhesion molecules), which are involved in endothelial cell contacts, and in beta-integrin 1 molecules, which are involved in integrin-mediated cell adhesion and signaling.

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