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Ryan D. McCarty, Jeffrey P. Mayer, Stephen N. Schwartz, Paul A. Knepper; Endogeneous Hyaluronidase and Hyaluronidase Inhibitors in POAG Aqueous. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2430.
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Hyaluronic acid (HA) plays a pivotal role in cell differentiation, cell signaling and aging. The concentration of HA is a balance of synthesis by hyaluronic synthetase and degradation by one of six hyaluronidases. The purpose of this study was to determine if hyaluronidase-2 and hyaluronidase inhibitors are present in aqueous.
Normal and primary open-angle glaucoma (POAG) aqueous were analyzed. Western blots were performed using a rabbit polyclonal 1° anti-hyaluronidase-2 antibody (Santa Cruz; 1:500) and a goat anti-rabbit 2° antibody (Jackson;1:3000). HA zymography was used to detect hyaluronidase in aqueous. Gels were first subjected to polyacrylamide gel electrophoresis, then transfered to a hyaluronidase buffer (pH 7.4) for 16 hours and finally stained in stages with Alcian Blue and Coomassie Blue respectively. Reverse HA zymography was used to detect hyaluronidase inhibitors by incubating the gels with 0.5 units/mL of bovine testicular hyaluronidase-containing solution for 16 hours, subjected to 0.1 mg/mL Pronase E for 4 hours and analyzed.
Western blot analysis showed a hyaluronidase-2 at 54 kDa detectable in 1 ug protein of aqueous. HA zymogram results showed a detectable band at 54 kDa. The reverse HA zymogram displayed a hyaluronidase inhibitor at approximately 83 kDa detectable in 1 ug protein of aqueous. Densimetric analysis indicated that there was more relative staining for hyaluronidase in POAG than normal aqueous, and more relative staining for hyaluronidase inhibitor in normal than POAG aqueous.
This is the first identification of both hyaluronidase-2 and a hyaluronidase inhibitor in both normal and POAG aqueous. Our results demonstrate a modest increase in hyaluronidase-2 and modest decrease hyaluronidase inhibitors in POAG aqueous compared to normal aqueous. A change in the balance of hyaluronidase and its inhibitor in aqueous may be an important factor in the homeostasis of HA and the function of the trabecular meshwork.
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