sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

Michael Giovingo, Michael Nolan, Ryan McCarty, Iok Hou Pang, Abbot F. Clark, Rachel M. Beverley, Steven Schwartz, W. Daniel Stamer, Loyal Walker, Algis Grybauskas, Kevin Skuran, Paulius V. Kuprys, Beatrice Y.J.T. Yue, Paul A. Knepper

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Abstract

Purpose: CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is signifcantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outfow resistance in vitro. Methods: Adenoviral constructs of human standard CD44 (Ad-CD44S), soluble CD44 (Ad-sCD44), and empty viral cDNA were injected into the vitreous of BALB/cJ mice, followed by serial IOP measurements. Overexpression of CD44S and sCD44 was verifed in vitro by enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Anterior segments of porcine eyes were perfused with the isolated sCD44. sCD44-treated human trabecular meshwork (TM) cells and microdissected porcine TM were examined by confocal microscopy and Optiprep density gradient with western blot analysis to determine changes in lipid raft components. Results: Intravitreous injection of adenoviral constructs with either Ad-CD44S or Ad-sCD44 vectors caused prolonged ocular hypertension in mice. Eight days after vector injection, Ad-CD44S signifcantly elevated IOP to 28.3±1.2 mmHg (mean±SEM, n=8; p<0.001); Ad-sCD44 increased IOP to 18.5±2.6 mmHg (n=8; p<0.01), whereas the IOP of uninjected eyes was 12.7±0.2 mmHg (n=16). The IOP elevation lasted more than 50 days. Topical administration of a γ-secretase inhibitor normalized Ad-sCD44-induced elevated IOP. sCD44 levels were signifcantly elevated in the aqueous humor of Ad-CD44S and Ad-sCD44 eyes versus contralateral uninjected eyes (p<0.01). Anterior segment perfusion of isolated 32-kDa sCD44 signifcantly decreased aqueous outfow rates. Co-administration of isolated sCD44 and CD44 neutralizing antibody or of γ-secretase inhibitor signifcantly enhanced fow rates. sCD44-treated human TM cells displayed cross-linked actin network formation. Optiprep density gradient and western blot analysis of human TM cells treated with sCD44 showed decreased annexin 2 expression and increased phosphorylated annexin 2 and caveolin 1 expression. Conclusions: Our data suggest that sCD44 increases outfow resistance in vivo and in vitro. Viral overexpression of both CD44S and sCD44 is suffcient to cause ocular hypertension. Infusion of sCD44 in porcine anterior segment eyes signifcantly decreased flow rates. Notably, sCD44 enhanced cross-linked actin network formation. The elevated sCD44 levels seen in POAG aqueous humor may play an important causative role in POAG pathogenesis.

Original languageEnglish
Pages (from-to)2151-2164
Number of pages14
JournalMolecular vision
Volume19
StatePublished - 1 Nov 2013

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Giovingo, M., Nolan, M., McCarty, R., Pang, I. H., Clark, A. F., Beverley, R. M., Schwartz, S., Stamer, W. D., Walker, L., Grybauskas, A., Skuran, K., Kuprys, P. V., Yue, B. Y. J. T., & Knepper, P. A. (2013). sCD44 overexpression increases intraocular pressure and aqueous outflow resistance. Molecular vision, 19, 2151-2164.