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K. E. Keller, J. M. Bradley, M. J. Kelley, T. S. Acott; Effect of Modifiers of Glycosaminoglycan Biosynthesis on Outflow Facility in Perfusion Culture. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2052.
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© ARVO (1962-2015); The Authors (2016-present)
Glycosaminoglycans (GAGs) have long been implicated as a source of outflow resistance in intraocular pressure (IOP) regulation. Previous studies have investigated the role of GAG degradation on outflow facility. The current study utilizes sodium chlorate, an inhibitor of GAG sulfation, and xylosides, which provide a competitive nucleation point for new GAG biosynthesis, to further study the roles of GAGs in outflow resistance.
Outflow facility was measured in perfused porcine anterior segment organ culture. After an initial stabilization period, 20 mM or 50 mM sodium chlorate, 1 mM p-nitrophenyl-alpha-D-xylopyranoside (αPNP; control isomer) or p-nitrophenyl-beta-D-xylopyranoside (ßPNP) were constantly perfused for 48 hours. Anterior segments were formalin-fixed and embedded in paraffin. Perturbation of several extracellular matrix (ECM) components was investigated by immunofluorescence and confocal microscopy. Parallel experiments were conducted on cultured TM cells and ECM components were analyzed by Western blotting and immunofluorescence.
Treatment with either 20 mM chlorate or ßPNP increased outflow facility approximately 3-fold over 48 hours. However, treatment with 50 mM chlorate slightly decreased outflow, while αPNP did not significantly change flow rate. The outflow increase with 20 mM chlorate was reversed by addition of 10 mM sodium sulfate. Microscopic analysis of anterior segments treated with chlorate or ßPNP showed altered patterns of immunostaining of several ECM molecules, including fibronectin, fibromodulin and decorin. When cultured TM cells were treated with ßPNP or chlorate, tenascin C deposition into the ECM was severely reduced, although fibronectin was not substantially affected. Decorin expression increased with ßPNP and chlorate treatments, labeling in a punctuate perinuclear pattern. Conversely, fibromodulin expression decreased, especially with chlorate treatments.
Two different modifiers of GAG biosynthesis were found to increase outflow facility and affect expression of several ECM components. This study highlights the importance of GAGs in ECM organization in normal TM and provides further support for a crucial role of functional GAGs in the regulation of outflow facility.
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