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Shinwu Jeong, Aditi Bauskar, Wendy J Mack, Jerome Mauris, Pablo Argueso, Noorjahan A Panjwani, Stephen C Pflugfelder, M Elizabeth Fini; Clusterin (CLU) Prevents Ocular Surface (OCS) Barrier Damage by an All-or-None Mechanism in a Preclinical Mouse Model of Dry Eye. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):345.
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© ARVO (1962-2015); The Authors (2016-present)
CLU, a glycoprotein found in tears, inhibits MMP-9 activity that is causative of OCS barrier damage due to desiccating stress (DS). We showed previously that topical CLU protects against DS-induced damage (ARVO2014). Here we studied mechanism.
DS was created in C57BL/6J female mice by application of the scopolamine plus air draft model (Dursen et al., IOVS, 2002) for 5 days. For a moderate DS challenge, we omitted SH injection. Natural or recombinant CLU in PBS was applied topically in 1 µl, 4 times daily. OCS barrier function was quantified by measuring permeability to Fluoresoft®-0.35% ophthalmic dye (FS). CLU and LGALS3 binding assay was performed in vitro, and protection of LGALS3 cleavage, in vivo. CLU in tears was quantified by ELISA.
In dose-response experiments, CLU treatment, but not BSA), either blocked FS uptake completely (1 µg/ml), or had no effect (0.6 µg/ml), i.e., an all-or-none response with a sharp transition (p<0.0001). However, under moderate DS, a dose response was observed in recovery from the damage: 0.1 µg/ml CLU was as effective as 1 µg/ml, however, a partial effect was observed at 0.01 µg/ml. Topical CLU protected the barrier molecule LGALS3 against cleavage. CLU protein bound to LGALS3-beads and was eluted by lactose (an LGALS3 counter-ligand), but not by sucrose, indicating a specific association via the LGALS3 carbohydrate-binding domain. The mean CLU concentration in tears of mice kept under ambient conditions was 5.2±0.4 µg/ml. This was reduced to 3.7±0.3 µg/ml in mice subjected to the 5-day DS protocol, i.e., ~30% reduction. Heterozygous CLU+/- KO mice have half the CLU gene dose of wild-type (WT) mice and we determined they also have ~half the mean tear CLU concentration, 2.15±0.6 µg/ml (p=2.1x10-5; n=7). After moderate DS challenge for 4 weeks, FS uptake in WT eyes was ~3x greater than baseline controls. In contrast, the uptake in CLU+/- KO mice was ~10x higher (p<0.0001; n=4).
In mice subjected to DS, topical CLU protects the OCS barrier against proteolysis via a remarkable all-or-none mechanism that may involve CLU binding to LGALS3 and intercalation into the barrier. Reduced levels of CLU in tears cause barrier vulnerability to DS. Thus, topical CLU application may be an effective therapeutic approach to prevent barrier damage in DE.
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