Segmental Versican Expression in the Trabecular Meshwork and Involvement in Outflow Facility

Abstract

PURPOSE. Versican is a large proteoglycan with numerous chondroitin sulfate (CS) glycosaminoglycan (GAG) side chains attached. To assess versican's potential contributions to aqueous humor outflow resistance, its segmental distribution in the trabecular meshwork (TM) and the effect on outflow facility of silencing the versican gene were evaluated. METHODS. Fluorescent quantum dots (Qdots) were perfused to label outflow pathways of anterior segments. Immunofluorescence with confocal microscopy and quantitative RT-PCR were used to determine versican protein and mRNA distribution relative to Qdot-labeled regions. Lentiviral delivery of shRNAsilencing cassettes to TM cells in perfused anterior segment cultures was used to evaluate the involvement of versican and CS GAG chains in outflow facility. RESULTS. Qdot uptake by TM cells showed considerable segmental variability in both human and porcine outflow pathways. Regional levels of Qdot labeling were inversely related to versican protein and mRNA levels; versican levels were relatively high in sparsely Qdot-labeled regions and low in densely labeled regions. Versican silencing decreased outflow facility in human and increased facility in porcine anterior segments. However, RNAi silencing of ChGn, an enzyme unique to CS GAG biosynthesis, increased outflow facility in both species. The fibrillar pattern of versican immunostaining in the TM juxtacanalicular region was disrupted after versican silencing in perfusion culture. CONCLUSIONS. Versican appears to be a central component of the outflow resistance, where it may organize GAGs and other ECM components to facilitate and control open flow channels in the TM. However, the exact molecular organization of this resistance appears to differ between human and porcine eyes. 1,2 Much of this resistance resides within the trabecular meshwork (TM) putatively within 7 to 14 m of the inner wall of Schlemm's canal in a region known as the juxtacanalicular (JCT) or cribriform region. 1-4 Since the 1950s, involvement of extracellular matrix (ECM) in outflow resistance has commonly been evoked. 1,2,5-10 There is also considerable evidence of a direct contribution to the resistance by some cell populations within this region. 1,3 More recently, synergistic interaction between the JCT and Schlemm's inner wall endothelial cells has been suggested. 2,6,12-14 Although perfusion of GAG-degrading enzymes reduces the outflow resistance in numerous species, in humans and primates, this effect has been controversial. 17-21 The identity of the specific GAGs, proteoglycans, or other ECM components that comprise outflow resistance remains unclear. 2 Both conceptually and based on several observations, versican, with supportive contributions from its attached chondroitin sulfate (CS) GAG chains and hyaluronan (HA) interactions, seems a likely contributor. 2,24 Up to 23 CS GAG side chains can be attached to these two central domains. These CS chains appear to extend away from the core protein in all directions, thus minimizing electrostatic interactions and filling large hydrodynamic volumes. Conceptually, this design is ideal to regulate movement of aqueous humor through the TM. 27 Mechanical stretching or distortion appears to be the mechanism by which TM cells sense elevated IOP and trigger IOP homeostatic responses. 30 -33 TNF␣ and IL-1␣, both of which increase outflow facility, also produce changes in versican mRNA level and isoform distribution similar t