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AM Miller, J Choi, PA Knepper; Does the Vitreous Humor Act as a Reservoir for Cytotoxic sCD44? . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1033.
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Purpose: CD44 is a hyaluronic acid (HA) binding protein and cell receptor which is shed from the cell surface as soluble CD44 (sCD44). sCD44 is increased in the aqueous humor in primary open-angle glaucoma (POAG) and adversely affects the viability of bovine trabecular meshwork cells. Since the vitreous humor contains large amounts of HA, we determined the sCD44 concentration in vitreous humor. Methods: Liquid and cortical vitreous was removed from post-mortem donor eyes within 24 h; vitreous samples were subjected to liquification at 85ºC for 20 min; 100 µl aliquots were incubated with extraction buffer (20 mM Tris, pH 7.5, 0.15 M NaCl, 1% Triton X-100, and protease inhibitors) for 2 h at 4ºC, followed by centrifugation, and then re-solubilized in 0.5% deoxycholate and 0.1 % sodium dodecyl sulfate for 2 h at 4ºC, followed by centrifugation. After each centrifugation, the supernatant was collected and 1 µg protein equivalent was analyzed using an ELISA according to the manufacturer's instructions. Results: The ELISA revealed that ≷95% of sCD44 positive material in vitreous was present in the Triton X-100-soluble fraction. sCD44 was identified by Western blot. sCD44 was three-fold greater in liquid vitreous as compared to aqueous humor. Moreover, increased pressure decreased sCD44 binding to HA in HA affinity columns. Conclusion: Vitreous contains large amounts of sCD44; the amount of sCD44 increased with aging. These results suggest vitreous acts as a reservoir for cytotoxic sCD44. Because sCD44 is detrimental to cell survival and sCD44 accumulates in vitreous with aging, diffusion of sCD44 from the vitreous to susceptible cells could contribute to the cell loss in POAG and in aging.
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