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M Elizabeth Fini, Aditi Bauskar, Shinwu Jeong; Clusterin (CLU) Prevents Ocular Surface Damage in a Mouse Model for Human Dry Eye Disease. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3060.
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© ARVO (1962-2015); The Authors (2016-present)
MMP9 is a key mediator of ocular surface damage due to dry eye. CLU is an extracellular chaperone protein with cytoprotective and anti-inflammatory properties, expressed at high levels by the corneal epithelium. We recently reported that CLU inhibits the proteolytic activity of MMP9 and inhibits MMP9-mediated damage to epithelial barrier function. We further found that expression of CLU in the corneal epithelium was greatly reduced under dry eye conditions (Am J Pathol 180:2028, 2012). We hypothesized that supplementation of CLU levels would protect the ocular surface against the damaging effects of dry eye.
Desiccating stress was created in female C57BL/6J mice (6-8 weeks) using the air draft plus scopolamine protocol. Air draft was applied to perforated cages for 18 hours daily with 30-40% humidity at 80 degrees F. Scopolamine hydrobromide (0.5 mg/0.2 mL in PBS) was injected subcutaneously, 4 times daily for 5 days or 7 days. At the same time, 1 ul of recombinant human sCLU (secreted form, R&D Systems), formulated at 1 or 10 ug/ml in PBS, was applied topically to the ocular surface 4 times daily. Corneal epithelial barrier function was quantified by measuring permeability to carboxyfluorescein (CBF) dye. Results were compared to baseline controls treated with PBS alone. An immortalized human corneal limbal epithelial (HCLE) cell line (I. Gipson, Harvard) was used for MMP9 expression studies. Secreted MMP9 was visualized by zymography and relative band density was quantified using Image J software. Statistical significance was determined by t-test.
CBF uptake in eyes stressed for 5 days while also being treated with PBS alone was 17-fold greater than baseline (P<10E-7, n=4). In contrast, CBF uptake in eyes stressed for 5 days while also being treated with sCLU was only 1.7-fold (1 ug/ml) or 1.4-fold (10 ug/ml) greater than baseline (P<10E-6, n=4). These results were reproduced in a second experiment utilizing 7 days of stress (P<10E-9, n=9). Treatment of HCLE cells with sCLU (40 ug/ml) significantly reduced the stimulatory effects of TNF-alpha (5 ng/ml) on MMP9 (P<0.01, n=3), but not of TGF-beta (5 ng/ml). In contrast, sCLU alone, or a BSA control, had no effect.
Topical CLU can protect the ocular surface against the damaging effects of desiccating stress, which models human dry eye disease. CLU inhibits TNF-alpha stimulated MMP-9 expression.
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