Effects of MMP–10 and a Cathepsin Inhibitor, Cystatin C, on Organ–Cultured Normal and Diabetic Human Corneas

Abstract

Abstract: : Purpose: We have recently documented an increased expression of matrix metalloproteinase–10 (MMP–10) and cathepsin F in the epithelium of human corneas from patients with diabetes and diabetic retinopathy (DR). The purpose of this work was to (1) examine the action of MMP–10 on wound healing and DR marker expression in organ–cultured normal corneas, and (2) determine the effects of cathepsin inhibition on organ cultured corneas from DR patients. Methods: Human organ–cultured corneas were wounded using n–heptanol to preserve epithelial basement membrane (BM). Purified MMP–10 was added to air–exposed top of normal wounded corneas at 1 µg/ml in 20 µl, 1–2 times a day until wound closure. Wounded DR corneas were treated with broad–spectrum MMP inhibitors, galardin at 5 µM, or a chemically modified tetracycline, CMT–3 (from Collagenex), at 50 µg/ml, in medium. Some DR corneas were also treated with a cathepsin inhibitor, cystatin C, at 50 µg/ml. After wound healing was complete, corneas were cultured for another week and processed for immunohistochemical analysis of diabetic markers, epithelial integrin α₃ß₁, and α5 chain of BM component, laminin–10. Results: Analysis of 26 normal corneas showed that epithelial defects healed on average by 3.4±0.8 (mean±SEM) days without MMP–10 and by 5.5±1.9 days with MMP–10 present. In MMP–10–treated corneas, immunofluorescent staining for laminin α5 chain was discontinuous in the epithelial BM, and integrin α₃ß₁ staining in the epithelium was weak and disorganized, very similar to DR corneas. Compared to vehicle treatment, galardin or CMT–3 did not show any effect on healing of organ–cultured and wounded DR corneas. Possibly, these MMP inhibitors also blocked other MMPs that are needed for corneal wound healing, e.g., MMP–9. Cystatin C treatment reduced cathepsin F staining in DR corneal epithelium. Moreover, integrin α₃ß₁ staining increased and became more regular, similar to normal corneas. This result was obtained in all three pairs of DR corneas analyzed. Wound healing rates, however, were not significantly different with or without cystatin C treatment. Conclusions: The data support the hypothesis that increased activity of certain proteinases may be responsible for clinically observed diabetic corneal abnormalities. Specific inhibition of proteinase expression using antisense oligonucleotides or siRNA may be an effective way to restore normal wound healing and marker patterns in corneas of diabetic and DR patients.

Keywords: cornea: epithelium • diabetes • proteolysis