Yuichi Uchino1, Jerome Mauris1, Ashley M Woodward1, Julia Dieckow1, Francisco Amparo1, Reza Dana1, Flavio Mantelli2, Pablo Argüeso3. 1. Schepens Eye Research Institute and Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts. 2. Campus Bio Medico University of Rome, Rome, Italy. 3. Schepens Eye Research Institute and Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts. Electronic address: pablo_argueso@meei.harvard.edu.
Abstract
PURPOSE: To investigate the expression, release, and proteolytic degradation of galectin-3 in patients with dry eye disease. DESIGN: Observational case series with a comparison group. METHODS: Tear washes and conjunctival impression cytology specimens were collected through standard procedures from 16 patients with dry eye and 11 age-matched healthy subjects. Galectin-3 content in tears was analyzed by quantitative Western blot, using recombinant galectin-3 protein to generate a calibration curve. The relative expression of galectin-3 and matrix metalloproteinase 9 (MMP9) was evaluated by quantitative polymerase chain reaction. The cleavage of galectin-3 was studied in vitro using activated recombinant MMP9 and protease inhibitors. RESULTS: The concentration of galectin-3 protein in tears, but not galectin-3 expression in conjunctival epithelium, was significantly higher in tears of patients with dry eye (0.38 ng/μg total protein, range 0.04-1.36) compared to healthy subjects (0.12 ng/μg total protein, range 0.00-0.41) (P < .01). By Western blot, an intact (∼28.0 kDa) galectin-3 band was identified in tear samples from healthy subjects, whereas 50% of the dry eye samples were characterized by the additional presence of a partially degraded form (∼25.4 kDa). In our experiments, elevated expression of MMP9 in dry eye subjects correlated with the ability of active MMP9 to cleave galectin-3 from recombinant origin. Interestingly, cleavage of endogenous galectin-3 in tear samples was impaired using a broad-spectrum proteinase inhibitor cocktail, but not the pan-specific MMP inhibitor GM6001, suggesting the presence of proteases other than MMPs in promoting galectin-3 degradation in dry eye. CONCLUSIONS: Our results indicate that release of cellular galectin-3 into tears is associated with epithelial dysfunction in dry eye, and that galectin-3 proteolytic cleavage may contribute to impaired ocular surface barrier function.
PURPOSE: To investigate the expression, release, and proteolytic degradation of galectin-3 in patients with dry eye disease. DESIGN: Observational case series with a comparison group. METHODS: Tear washes and conjunctival impression cytology specimens were collected through standard procedures from 16 patients with dry eye and 11 age-matched healthy subjects. Galectin-3 content in tears was analyzed by quantitative Western blot, using recombinant galectin-3 protein to generate a calibration curve. The relative expression of galectin-3 and matrix metalloproteinase 9 (MMP9) was evaluated by quantitative polymerase chain reaction. The cleavage of galectin-3 was studied in vitro using activated recombinant MMP9 and protease inhibitors. RESULTS: The concentration of galectin-3 protein in tears, but not galectin-3 expression in conjunctival epithelium, was significantly higher in tears of patients with dry eye (0.38 ng/μg total protein, range 0.04-1.36) compared to healthy subjects (0.12 ng/μg total protein, range 0.00-0.41) (P < .01). By Western blot, an intact (∼28.0 kDa) galectin-3 band was identified in tear samples from healthy subjects, whereas 50% of the dry eye samples were characterized by the additional presence of a partially degraded form (∼25.4 kDa). In our experiments, elevated expression of MMP9 in dry eye subjects correlated with the ability of active MMP9 to cleave galectin-3 from recombinant origin. Interestingly, cleavage of endogenous galectin-3 in tear samples was impaired using a broad-spectrum proteinase inhibitor cocktail, but not the pan-specific MMP inhibitor GM6001, suggesting the presence of proteases other than MMPs in promoting galectin-3 degradation in dry eye. CONCLUSIONS: Our results indicate that release of cellular galectin-3 into tears is associated with epithelial dysfunction in dry eye, and that galectin-3 proteolytic cleavage may contribute to impaired ocular surface barrier function.
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Authors: Mark D P Willcox; Pablo Argüeso; Georgi A Georgiev; Juha M Holopainen; Gordon W Laurie; Tom J Millar; Eric B Papas; Jannick P Rolland; Tannin A Schmidt; Ulrike Stahl; Tatiana Suarez; Lakshman N Subbaraman; Omür Ö Uçakhan; Lyndon Jones Journal: Ocul Surf Date: 2017-07-20 Impact factor: 5.033
Authors: Takazumi Taniguchi; Ashley M Woodward; Paula Magnelli; Nicole M McColgan; Sylvain Lehoux; Sarah Melissa P Jacobo; Jérôme Mauris; Pablo Argüeso Journal: J Biol Chem Date: 2017-05-09 Impact factor: 5.157