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Jaclyn Bermudez, Abbot F Clark, Weiming Mao; Identification of Proteins Involved in the Formation of Cross-Linked Actin Networks in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5680. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is a leading cause of blindness worldwide, and the primary risk factor of glaucoma is increased intraocular pressure (IOP). In glaucoma patients, outflow resistance through the trabecular meshwork (TM) is abnormally elevated. Comparison between normal and glaucomatous TM cells and tissues revealed excessive formation of cross-linked actin networks (CLANs). CLANs are web-like polygonal structures found within confluent TM cells and TM tissues in situ. Glaucoma-associated factors such as TGFβ2 and dexamethasone (DEX) also induce CLAN formation. CLANs alter TM cell functions and may increase cell rigidity thereby contributing to elevated aqueous outflow resistance as well as IOP. However, the proteins involved in CLAN formation in confluent TM cells are not fully characterized. Therefore, we used a proteomic approach to identify the proteins that are involved in CLAN formation.
We treated confluent primary human TM cell cultures with 1% ethanol (EtOH) as vehicle control, 100 nM DEX, or 5 ng/ml TGFβ2 plus 1% (EtOH) for 7 to 10 days to induce CLAN formation. To enrich CLANs, we treated cells with 0.2% Triton-X-100 in ISO buffer to remove soluble proteins. The residual insoluble cytoskeletal proteins were collected and separated by 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) (Applied Biomics). Proteins with increased expression levels in both DEX and TGFβ2 treated samples compared to control cells were trypsin digested and subjected to mass spectrometry (MS) analysis. Differential expression of proteins identified by MS was confirmed by Western immunoblotting (WB) and immunocytochemistry (IHC) analysis.
A number of proteins were identified as being enriched in CLAN forming TM cells by MS analysis. Using WB and IHC, caldesmon, calponin, myosin light chain, and tropomyosin were found to be upregulated with DEX and TGFβ2 treatment and each co-localized with CLANs.
We identified a set of proteins that are differentially expressed in CLAN-enriched human TM cells. The potential role of these proteins in CLAN formation will be further investigated. These proteins may provide new insights into the pathogenesis of glaucoma.
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