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Janice A. Vranka, Kate E. Keller, Ted S. Acott; Structural Consequences of Versican Silencing on the Outflow Resistance Pathway and in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3263.
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Versican is a component of the extracellular matrix (ECM) of the trabecular meshwork (TM) and is a major contributor to the outflow resistance and the normal homeostatic response to elevated intraocular pressure (IOP). Versican is known to interact with a wide range of extracellular and cell-associated molecules and is thought to exert its influence through complex protein-protein interactions in the TM. The purpose of this work was to determine which ECM components in the juxtacanalicular region (JCT) of the TM co-localize with versican and which components are affected when versican expression is silenced.
Anterior segment perfusion culture was used to analyze flow rates in human donor eyes in vitro. Immunofluorescence with confocal microscopy and 3D-image analysis using Imaris software were used to visualize protein distribution patterns. Pearson’s Correlation Coefficients were obtained to quantitate the amount of overlapping localization of two proteins. Lentiviral delivery of a versican shRNA-silencing vector to TM cells in perfused anterior segment cultures was used to evaluate the extracellular architecture in the JCT in the absence of newly-synthesized versican. Primary cultured human TM cells were grown in the absence and presence of the versican shRNA silencing vector and ECM gene transcript levels were determined by quantitative RT-PCR.
Immunostaining for versican and fibrillin-1, and versican and microfibrillar associated protein-5 (MFAP-5) showed a high degree of overlap in the JCT region of the TM. Versican immunostaining also overlapped somewhat with SPARC in human TM tissues. Versican silencing was previously shown to decrease outflow facility in human anterior segment organ culture but the mechanism is unknown. Here it was shown to disrupt the fibrillar-like pattern of versican in the JCT, but had only a minimal effect on fibrillin-1 immunostaining in the same region. In cultured human TM cells, fibrillin-1, fibrillin-2, fibronectin, thrombospondin-1 and SPARC gene expression were all affected by versican-silencing at 72 hours, whereas tenascin C expression was minimally affected.
Versican is a central component of the outflow resistance and appears to interact with a select subset of ECM components within the JCT architecture. Versican silencing in both human TM tissue and primary cultured cells results in altered protein and gene expression patterns, respectively. Therefore versican-specific interactions may provide a structural context for an improved understanding of the overall outflow resistance mechanism in the homeostatic response to sustained IOP.
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