Matrix metalloproteinase-2 degrades fibrillin-1 and fibrillin-2 of oxytalan fibers in the human eye and periodontal ligaments in vitro

Abstract

Oxytalan fibers are distributed in the eye and periodontal ligaments (PDL). The ciliary zonule, known as Zinn's zonule, in the eye is composed of oxytalan fibers, which are bundles of microfibrils consisting mainly of fibrillin-1 and fibrillin-2. As turnover of oxytalan fibers is slow during life, their degradation mechanism remains unclarified. This study was performed to examine degradation pattern of fibrillin-1 and fibrillin-2 by experimental MMP activation. We cultured human non-pigmented ciliary epithelial cells (HNPCEC) and PDL fibroblasts for 7 days, then treated them with concanavalin A to activate matrix metalloproteinase (MMP)-2, and examined the degradation of fibrillin-1 and fibrillin-2 for 72 hr using immunofluorescence. At 7 days of HNPCEC culture, fibrillin-1-positive fibers were observed, some of which merged with fibrillin-2. After MMP-2 activation, fibrillin-1-positive fibers became thin and disappeared by 72 hr, while fibrillin-2-positive fibers disappeared almost completely within 24 hr. At 7 days of PDL fibroblast culture, fibrillin-1-positive fibers were mostly merged with fibrillin-2. After MMP-2 activation, fibrillin-1-positive fibers became thin by 24 hr and had almost disappeared by 48 hr, while fibrillin-2-positive fibers decreased constantly after 24 hr. A MMP-2 inhibitor completely suppressed these degradations. These results suggest that the patterns of fibrillin-1 and fibrillin-2 degradation differ between the eye and the PDL, possibly reflecting the sensitivity of fibrillin-1 and fibrillin-2 of each type of oxytalan fiber against MMP-2.

Keywords: ciliary zonule; fibrillin; microfibrils; oxytalan fibers.

Fig. 1

ConA-induced pro-MMP-2 activation. Human non-pigmented ciliary epithelial cells (A) and periodontal ligaments fibroblasts (B) were cultured for 7 days, and then various concentrations (0, 10, 50 ng/ml) of ConA were added to the medium for an additional 72 hr. Cell lysates obtained at 6 hr after addition of ConA were analyzed by zymography. Harvested samples from non-pigmented ciliary epithelial cells (lane 1) and periodontal ligament fibroblast (lane 2) obtained at 72 hr were similarly subjected to gelatin zymography (C). Pro-MMP-2: latent-MMP-2, 72 kDa; Int MMP-2: Intermediate-MMP-2, 64 kDa; Act MMP-2: Active-MMP-2, 62 kDa.

Fig. 2

Degradation of fibrillin-1 and fibrillin-2 by active MMP-2. (A) Double immunofluorescence for fibrillin-1 (upper panels) and fibrillin-2 (middle panels) in cultures of human non-pigmented ciliary epithelial cells. Human non-pigmented ciliary epithelial cells were cultured for 7 days, and then 50 ng/ml ConA was added to the medium for an additional 72 hr. Cells were then labeled simultaneously for fibrillin-1 (green) (upper panels), fibrillin-2 (red) (middle panels), and superimposition of both labels (lower panels) at 0, 24, 48 and 72 hr. Bar=20 µm. (B) Planimetry analysis of the positive staining signals was performed using the Image J program. The relative area of positive signals per unit area at 0 hr was calculated using Image J, with the control set as 1. Data represent the mean±S.D. (standard deviation) of four independent experimental determinations.

Fig. 3

Degradation of fibrillin-1 and fibrillin-2 by active MMP-2. (A) Double immunofluorescence for fibrillin-1 (upper panels) and fibrillin-2 (middle panels) in cultures of human periodontal ligament fibroblasts. Human periodontal ligament fibroblasts were cultured for 7 days, and then 50 ng/ml ConA was added to the medium for an additional 72 hr. Cells were then simultaneously labeled for fibrillin-1 (green) (upper panels), fibrillin-2 (red) (middle panels), and superimposition of both labels (lower panels) at 0, 24, 48 and 72 hr. Bar=20 µm. (B) Planimetry analysis of the positive signals was performed using the Image J program. The relative area of positive signals at 0 hr was obtained using Image J with the control set as 1. Data represent the mean±S.D. (standard deviation) of four independent experimental determinations.

Fig. 4

Evidence that MMP-2 degrades fibrillin-1 and fibrillin-2. Human non-pigmented ciliary epithelial cells (A) and periodontal ligament fibroblasts (B) were cultured for 7 days, and then 50 ng/ml ConA was added to the medium for an additional 72 hr. In some cultures, 2.5 µM MMP-2 inhibitor I as well as 50 ng/ml ConA was added to the medium. After 72 hr, the cells were simultaneously labeled for fibrillin-1 (green) (upper panels) and fibrillin-2 (red). The left panel is the control (non-treated) culture, the middle panel is the ConA-treated culture, and the right panel is the culture treated with both ConA and MMP-2 inhibitor I. Bar=20 µm.