Alteration of galectin-3 in tears of patients with dry eye disease

Yuichi Uchino, Jerome Mauris, Ashley M. Woodward, Julia Dieckow, Francisco Amparo, Reza Dana, Flavio Mantelli, Pablo Argüeso

Research output: Contribution to journalArticle

23 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)1027-1035.e3
JournalAmerican Journal of Ophthalmology
Volume159
Issue number6
DOIs
Publication statusPublished - 2015 Jun 1
Externally publishedYes

Fingerprint

Galectin 3
Eye Diseases
Tears
Matrix Metalloproteinase 9
Healthy Volunteers
Matrix Metalloproteinase Inhibitors
Peptide Hydrolases
Western Blotting
Proteins
Protease Inhibitors
Matrix Metalloproteinases
Calibration
Cell Biology

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Uchino, Y., Mauris, J., Woodward, A. M., Dieckow, J., Amparo, F., Dana, R., ... Argüeso, P. (2015). Alteration of galectin-3 in tears of patients with dry eye disease. American Journal of Ophthalmology, 159(6), 1027-1035.e3. https://doi.org/10.1016/j.ajo.2015.02.008

Alteration of galectin-3 in tears of patients with dry eye disease. / Uchino, Yuichi; Mauris, Jerome; Woodward, Ashley M.; Dieckow, Julia; Amparo, Francisco; Dana, Reza; Mantelli, Flavio; Argüeso, Pablo.

In: American Journal of Ophthalmology, Vol. 159, No. 6, 01.06.2015, p. 1027-1035.e3.

Research output: Contribution to journalArticle

Uchino, Y, Mauris, J, Woodward, AM, Dieckow, J, Amparo, F, Dana, R, Mantelli, F & Argüeso, P 2015, 'Alteration of galectin-3 in tears of patients with dry eye disease', American Journal of Ophthalmology, vol. 159, no. 6, pp. 1027-1035.e3. https://doi.org/10.1016/j.ajo.2015.02.008
Uchino, Yuichi ; Mauris, Jerome ; Woodward, Ashley M. ; Dieckow, Julia ; Amparo, Francisco ; Dana, Reza ; Mantelli, Flavio ; Argüeso, Pablo. / Alteration of galectin-3 in tears of patients with dry eye disease. In: American Journal of Ophthalmology. 2015 ; Vol. 159, No. 6. pp. 1027-1035.e3.
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AB - 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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