DETERMINATION OF PROTEINS INVOLVED IN THE FORMATION OF CROSS-LINKED ACTIN NETWORKS IN THE TRABECULAR MESHWORK

Purpose: Glaucoma is a leading cause of blindness worldwide and the primary risk factor of glaucoma is increased intraocular pressure (IOP). IOP is determined by the equilibrium of aqueous humor (a fluid that fills the anterior segment of the eye) production and outflow. In glaucoma patients, the outflow resistance through the trabecular meshwork (TM), a special tissue located at the angle between the cornea and iris, is abnormally elevated. Inside TM cells, actin proteins form cross-linked actin networks (CLANs). Excessive formation of these unique structures can be found in the glaucomatous TM. They can also be induced by dexamethasone (DEX) and TGFβ2. It is suggested that CLANs increase cell rigidity and therefore elevate aqueous humor outflow resistance. However, the proteins that are involved in CLANs formation are not fully identified. We hypothesize that by comparing CLANs enriched and un-enriched TM cells, we will be able to identify the proteins that are involved in CLANs formation. Methods: We treated cultured mouse TM cells with DEX (100 nM), TGFβ2, (5 ng/mL), or ethanol (as vehicle control), evaluated CLANs formation, and separated the cell proteins by 2D gel electrophoresis. Differentially expressed proteins were analyzed by the Redfin software, and gel spots were picked and assessed by spectrometry analysis. Western blotting was used to confirm 2D gel results. Results: We found a subset of proteins that are differentially expressed in CLANs-enriched TM cells, compared to non-enriched samples. Among these proteins, ferritin heavy chain, glial fibrillary acidic protein (GFAP), and integrin alpha V, were confirmed by mass spectrometry and Western immunoblot. Conclusions: We found a subset of proteins that are differentially expressed in CLANs-enriched mouse TM cells. Their contributions and involvements in CLANs formation and regulation of TM cell morphology and functions are being investigated. They may provide new insight of the pathogenesis of glaucoma.