Trypsin-mediated enzymatic degradation of type II collagen in the human vitreous.

PURPOSE: Aging of the vitreous body can result in sight-threatening pathology. One aspect of vitreous aging is liquefaction, which results from the vanishing of collagen fibrils. We investigated the possibility that trypsins are involved in vitreous type II collagen degradation. METHODS: Immunohistochemistry and western blotting were used for detecting and locating trypsin isoforms in the vitreous and retina of human donor eyes. The capability of the retina to produce these trypsins was analyzed with polymerase chain reaction. Whether the different trypsins degraded type II collagen was tested in vitro. The sizes of the in vitro induced type II collagen degradation products were compared to those present in the vitreous of human eyes of different ages. RESULTS: Trypsin-1 and trypsin-2 were detected in the vitreous. In the retina, messenger ribonucleic acid (mRNA) coding for trypsin-2, -3, and -4 was present. Using immunohistochemistry, trypsin-2 was detected in microglial cells located in the vitreous and the retina. All trypsin isoforms degraded type II collagen and produced degradation products of similar sizes as those present in the vitreous. CONCLUSIONS: Trypsin-1 and trypsin-2 appear to have a function in the degradation of vitreous type II collagen. They could therefore have a role in the development of vitreous liquefaction.

In vitro phagocytosis of collagens by immortalised human retinal Müller cells.

PURPOSE: This study is a first step to investigate phagocytosis of collagens by human retinal Müller cells, since Müller cells could be involved in remodelling of the vitreous and vitreoretinal interface in the human eye. METHODS: Müller cells in culture were exposed to 2.0 microm fluorescent latex beads coated with BSA and human types I, II, and IV collagen and to non-coated beads for 2, 12, 24, and 48 h. To influence phagocytosis, cytochalasin B and anti-integrin subunits (alpha1, alpha2, and beta1) were added to the cells. Phagocytosis was evaluated by flow cytometry, transmission electron microscopy (TEM) and confocal microscopy. RESULTS: Müller cells preferred to phagocytose beads coated with type II collagen compared with type IV collagen-, BSA- and non-coated beads. Phagocytosis of type I collagen-coated beads was intermediate. TEM and confocal microscopic evaluation confirmed phagocytosis of the beads. No significant differences were observed in phagocytosis of type II collagen-coated beads in the case of addition of cytochalasin B and anti-integrin subunits. Immunohistochemical analyses revealed that Müller cells were positive, under all tested circumstances, for vimentin and CRALBP. Less than 5% of the cells tested were GFAP positive. CONCLUSIONS: Our observations demonstrate that human Müller cells in culture prefer to phagocytose type II collagen. In contrast, the phagocytosis of type IV collagen is comparable with the control coatings. We speculate that the relatively limited collagen phagocytosis by Müller cells supports a possible role for Müller cells in the slow process of vitreoretinal remodelling in adult human eyes.