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A new signaling pathway in the trabecular meshwork is involved in glaucomatous ocular hypertension, believes the research team that discovered it. Altered Wnt signaling may be responsible for pathogenic elevated IOP.
Fort Lauderdale, FL-Using genomics, researchers have discovered a newly identified signaling pathway in the trabecular meshwork that regulates IOP and that appears to be involved in glaucomatous ocular hypertension, according to Abbot "Abe" F. Clark, PhD, speaking here at the annual meeting of the Association for Research in Vision and Ophthalmology.
Dr. Clark is senior director of glaucoma research, Alcon Research, Fort Worth, TX, and adjunct professor, cell biology and genetics, University of North Texas Health Science Center at Fort Worth. He and his colleagues have been searching for pathogenic pathways because the molecular mechanisms responsible for glaucomatous damage to the trabecular meshwork are poorly understood. Approaches to identifying these pathogenic pathways include molecular genetics, genomics, proteomics, and disease research, all comparing normal and glaucomatous cells and tissues. These approaches have led to some proposed glaucomatous pathogenic pathways, including mutant myocilin, TGF-beta/CTGF, BMP/gremlin, ELAM1/IL-1, cochlin, and cross-linked actin networks, or CLANs.
For this study, Dr. Clark and colleagues used differential gene expression comparing normal and glaucomatous trabecular meshwork cells but carried the process further by confirming the differential expression in multiple samples, then showing that the altered expression results in the phenotype of elevated IOP. Techniques used to look at gene expression included extraction of RNA from normal and glaucomatous cells and tissues, RNA differential display and sequencing of differentially expressed bands, and gene chips to profile global gene expression. They used quantitative polymerase chain reaction (qPCR) to confirm differential expression.
Differentially expressed gene
Through these tests, the investigators discovered a differentially expressed gene, sFRP1 (secreted frizzled related protein); sFRP1 is an endogenous inhibitor of the Wnt signaling pathway, which is involved in embryogenesis as well as in certain cancers.
They looked at six normal and six glaucomatous trabecular meshwork cells with qPCR to measure sFRP1 levels and found a four- to five-fold increased expression in the glaucomatous cells (p < 0.01). This finding was confirmed at the protein level with 13 normal and 10 glaucomatous trabecular meshwork cell lines. In this test, a 60% increase in sFRP1 protein expression was seen in the glaucomatous cells (p < 0.05).
"We are hypothesizing that sFRP1 blocks normal Wnt signaling in the trabecular meshwork, and that leads to elevated IOP," Dr. Clark said.
Three Wnt signaling pathways have been described. According to Dr. Clark, interaction occurs in the normal trabecular meshwork between Wnt, frizzled, and coreceptor LRP5 or LRP6. This leads to the build-up of the signaling molecule beta-catenin, which enters the nucleus to turn on gene expression.
"In glaucoma, we believe that elevated sFRP1 blocks this Wnt signaling, allowing activation of pathways that mark beta-catenin for proteolytic degradation, which turns off Wnt-regulated gene expression," Dr. Clark said.
Other components of the Wnt signaling pathway also are expressed in the trabecular meshwork cells and tissues, including a variety of Wnt, frizzled, sFRP, and beta-catenin.
Because beta-catenin is an important signaling mediator, looking at levels of this molecule in the presence or absence of sFRP1 can help determine whether a functional Wnt signaling pathway is present in the trabecular meshwork, Dr. Clark said. "If there is, there should be an inverse correlation between the level of sFRP1 and the level of beta-catenin."
Through Western immunoblot analysis of seven trabecular meshwork cell lines, he and his colleagues found evidence to support that hypothesis. Most cells that had high levels of sFRP1 had lower levels of beta-catenin. Conversely, cells that had higher levels of beta-catenin had lower levels of sFRP1.
To determine whether this correlation had a functional effect on IOP, Dr. Clark and colleagues used human anterior segments perfused in the absence or presence of recombinant sFRP1 to examine the effect on outflow facility as well as the impact of sFRP1 on beta-catenin expression in the trabecular meshwork.