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A. R. Shepard, M. J. Nolan, J. C. Millar, I. H. Pang, T. Luan, N. Jacobson, M. B. Wax, A. F. Clark, P. A. Knepper; CD44 Overexpression Causes Ocular Hypertension in the Mouse. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2880.
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CD44 plays major roles in multiple physiological processes including inflammation, phagocytosis, and cell survival pathways. Soluble CD44 (sCD44) is an ectodomain fragment of the cell-surface receptor CD44. sCD44 is cytotoxic to trabecular meshwork (TM) and retinal ganglion cells (RGC) cells in culture, and is elevated in primary open angle glaucoma (POAG) vs. non-POAG patient aqueous humor. sCD44 concentration in aqueous humor correlates with the extent of visual field loss in POAG. This study examined the effects of adenoviral overexpression of either full-length or ectodomain fragment of CD44 on mouse IOP for establishment of a rodent model of POAG.
Human CD44 transcript variant 4 (CD44v4) cDNA (Origene #SC128160; RefSeq NM_001001391) was subcloned into the pacAd5.CMV.K-N.pA (U Iowa GTVC) shuttle vector for adenovirus production. A sCD44 version was also created by the introduction of a stop codon at the end of exon 5 (A>T, nt 1101) using the QuickChange XL mutagenesis kit (Stratagene). Expression of full-length and soluble CD44 was verified in vitro by ELISA (Bender BMS209/2). BALB/cJ mice were injected intravitreally (one eye only per mouse) with 4.8E7 pfu adenovirus followed by IOP measurements (Tonolab). sCD44 levels in pooled mouse aqueous humor were assessed by ELISA.
Elevated levels of CD44 were detected in cell lysate and medium from primary normal human TM cells transduced with either Ad5.CMV.CD44v4 or Ad5.CMV.sCD44 virus. In two separate studies, transduction of mouse eyes with either CD44v4 or sCD44 viral expression vectors caused ocular hypertension. The baseline IOP of the mouse was approximately 12 mmHg. At 8 days after vector injection, Ad5.CD44v4 significantly elevated IOP to 28.3 ± 1.2 mmHg (mean ± SEM, n = 8; p < 0.001); Ad5.sCD44 increased IOP to 18.5 ± 2.6 mmHg (n = 8; p< 0.01), whereas the IOP of uninjected eyes at the same time point was 12.7 ± 0.2 mmHg (n = 16). The IOP elevation lasted more than 60 days. sCD44 levels were significantly elevated in the aqueous humor of Ad5.CMV.CD44v4 and Ad5.CMV.sCD44 eyes vs. paired uninjected eyes (p<0.01).
We have initiated the establishment of a rodent model of POAG by showing that viral overexpression of both full-length and truncated human CD44 variant 4 (sCD44) in mouse eyes is sufficient to cause ocular hypertension. Our data suggest that the elevated sCD44 levels seen in POAG vs. non-glaucoma aqueous humor are not just an epiphenomenon but may play an actual causative role in POAG pathogenesis.
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