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Vijaykrishna Raghunathan, Julia Benoit, Christopher J Murphy; Cell derived matrices: Inducing a simple twist of fate towards a glaucomatous phenotype. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2452.
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© ARVO (1962-2015); The Authors (2016-present)
Using mechanotransduction systems, cells translate the intrinsic properties of extracellular matrix (ECM) to intracellular signals to control gene transcription, protein expression, and cell behavior. A number of studies have been performed to characterize pathologic ECM, only few studies have investigated the impact that 'pathologic' ECM have on ‘normal’ cell behavior. Here, we report changes in ECM derived from cells of glaucomatous (GTM) and non-glaucomatous (HTM) donors, and what effect these ECM have on non-glaucomatous cell mechanics and gene/protein expression.
Primary TM cells (1oTM) from at least 3 human eye bank donor eyes [GTM HTM] were each plated on aminated glass cover slips, were cultured in 10-7M dex or vehicle control for 4 weeks. Next, the GTM and HTM cells were dissociated and the mechanics of the deposited ECM was measured by atomic force microscopy (AFM). Decellularized ECM was either (i) characterized by proteomics/immunocytochemistry, or (ii) used for subsequent cell culture. 1oTM cells from a single HTM donor (♂, 50 y.o, HTM50) was cultured on the ECM, derived from GTM/HTM cells described above, for 3 days. Subsequently, cell mechanics was determined by AFM, gene & protein expression by qPCR & shotgun proteomics, and organization by immunocytochemistry.
The matrix deposited by GTM cells was at least 2 fold greater than HTM cells for each treatment condition. ECM deposited by both HTM and GTM cells treated with Dex was 2-fold stiffer. Fibronectin was more intertwined and dense in GTM ECM and was exacerbated with Dex. HTM50 cell stiffness was increased when cultured on ECM derived from GTM donors in comparison with that derived from HTM donors or on glass. Increase in cell stiffness was accompanied by marked increases in Myocilin and αB-Crystallin expression but significantly decreased expression of Hsp27, CTGF and Serpine1 in comparison cells cultured on HTM-ECM or on glass. Further, HTM50 cells remodeled the matrices from normal cells to a greater degree than matrices derived from glaucomatous donors.
For the first time, we have demonstrated that, ECM derived from glaucomatous cells are not only stiffer, but strongly influence the phenotype of normal HTM cells by altering cell stiffness (cytoskeletal dynamics), and gene/protein expression. These results suggest that a diseased ECM could drive a normal cell towards a diseased phenotype.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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