Loss of cell-matrix cohesiveness after phagocytosis by trabecular meshwork cells.

PURPOSE: To investigate the response of trabecular meshwork cells to phagocytic events. METHODS: Cultured bovine trabecular meshwork cells were established and exposed to latex microspheres for 40 to 44 hours. After phagocytosis, the cohesiveness of cells to their underlying matrix was measured by the susceptibility to trypsin, as indicated by the time needed to be liberated from culture plates. The amounts of two cell attachment proteins, fibronectin and laminin, in both the phagocytically challenged and the control cultures were measured at various postphagocytosis time points with an enzyme-linked immunosorbent assay. The fibronectin and laminin network was visualized with immunostaining. The mRNA levels were analyzed by Northern blot. Zymography using gelatin-containing gels was also performed to examine the gelatinase activities. RESULTS: Compared with controls, cells in phagocytically challenged cultures were more sensitive to trypsin. At the 4- and 8-hour postphagocytosis time points, the trypsinization time needed to suspend cells from tissue culture plates was significantly shorter for phagocytically challenged cells. Also, at these two time points, reduced amounts of fibronectin and laminin, as well as disruption of the fibronectin-laminin network, were observed in the phagocytically challenged trabecular meshwork cultures. The mRNA level for fibronectin was reduced, and a slightly increased gelatinase activity was noted. The fibronectin and laminin levels returned to normal by 24 hours. CONCLUSIONS: Results suggest that after phagocytosis, trabecular meshwork cells exhibit a short-term loss in cell-matrix cohesiveness. Such a loss may be related to diminished levels of cell attachment proteins.

Lysosomal enzyme and inhibitor levels in the human trabecular meshwork.

PURPOSE: To examine in the human trabecular meshwork lysosomal enzymes and one inhibitor of serine proteases that actively participate in the degradation of macromolecules into low molecular weight constituents. METHODS: Using an avidin-biotin-peroxidase technique, lysosomal proteases and alpha 1-proteinase inhibitor were examined in the trabecular meshwork of 23 human eyes with donor ages ranging from 2 to 90 years. These eyes were categorized into three age groups (< or = 20, 21 to 49, and > or = 50 years). Histochemical staining for lysosomal hydrolases was also performed on frozen sections of 20 human eyes. The staining was analyzed by an image analyzer and the levels of lysosomal proteases were further measured by biochemical assays. RESULTS: The trabecular meshwork from all the eyes stained intensely against antibodies to cathepsins B and G and alpha 1-proteinase inhibitor. The staining for elastase was weaker but evident. Image analyses revealed that the staining intensity for each protease or inhibitor was similar in all age groups. The staining in the uveal meshwork appeared to be the strongest among all the trabecular meshwork regions. Biochemical assays of tissue extracts confirmed that the enzyme and inhibitor levels were comparable among the three donor age groups. Activities of two lysosomal hydrolases, acid phosphatase and acid esterase, were also found in trabecular meshwork cells of 20 eyes. No apparent difference in enzyme activities was found with increasing age, and variation related to region was not observed. CONCLUSIONS: This study demonstrated the age-independent distribution of a variety of lysosomal enzymes and a protease inhibitor in the human trabecular meshwork. The presence of these proteins suggests a possible role in the metabolic operation of the trabecular meshwork.

Effects of antiglaucoma medications on bovine trabecular meshwork cells in vitro.

Using an in vitro culture system, we investigated the effects of five antiglaucoma drugs on growth and morphologic characteristics of bovine trabecular meshwork cells. Epinephrine hydrochloride (55-550 microM) and pilocarpine hydrochloride (0.8-16 mM), when added to the cultures for 3 days, inhibited trabecular cell growth in a dose-dependent manner. The lowest concentration at which the inhibitory effect was observed was 109 microM and 0.8 mM, respectively, for epinephrine and pilocarpine. Dipivefrin hydrochloride (26-260 microM), timolol maleate (116-1160 microM), and levobunolol hydrochloride (150-1500 microM) were also added to the cells for 3 days. These drugs caused a reduction in cell density, respectively, at concentrations higher than 103, 460, and 616 microM. Cell elongation was seen in cultures treated with epinephrine and dipivefrin, whereas levobunolol and timolol induced the cells to adopt a rounded appearance. Cells that had been exposed to pilocarpine were enlarged with numerous vacuoles. By scanning electron microscopic techniques, epinephrine, timolol, and levobunolol were found to retard the phagocytosis of latex beads by trabecular meshwork cells. Immunostaining with the use of antibodies to vimentin and actin revealed disorganization and condensation of cytoskeletal fibers in trabecular meshwork cells after treatment with epinephrine and dipivefrin. Little change was seen with comparable concentrations of a preservative, benzalkonium chloride, and a vehicle, Liquifilm tears. These results showed that antiglaucoma drugs, depending on their concentrations, may profoundly influence the growth and activity of trabecular meshwork cells.

Expression of modified low-density lipoprotein receptors by trabecular meshwork cells.

We examined the incorporation of fluoresceinated low-density lipoprotein (LDL) and acetylated or acetoacetylated low-density lipoprotein (A-LDL or AA-LDL) by a number of ocular cells in culture. All the cells investigated, including bovine, monkey, human trabecular meshwork cells, human corneal endothelial cells, human corneal stromal cells and human scleral cells, took up fluorescently labeled LDL. The bovine, monkey and human trabecular meshwork cells showed the strongest fluorescence reactions. In addition, we found that the trabecular meshwork cells became fluorescent after incubations with labeled A-LDL or AA-LDL. They were the only cell type examined that possessed this capacity. The fluorescence intensity was markedly diminished by adding to the incubation solution either fucoidin, a competitive inhibitor of modified LDL uptake, unlabeled A-LDL or AA-LDL. The trabecular meshwork cells in situ also became brightly labeled after exposure to fluoresceinated native LDL, A-LDL or AA-LDL. The uptake of modified LDL separated the trabecular meshwork cells from other types of ocular cells, which may be used to aid identification of trabecular meshwork cells in culture as well as in situ. This property also suggested that trabecular meshwork cells may have some functional similarities to macrophages.

Effects of chondroitin sulfate on cultured human retinal pigment epithelial cells.

We investigated the effects of chondroitin sulfate on growth, morphology and ultrastructure of retinal pigment epithelial (RPE) cells in culture. When added to the medium, chondroitin sulfate reduced cell density in RPE cultures in a dose-dependent manner. Compared with the controls, the treated cells appeared to be larger and more granular. Electron microscopic examinations revealed accumulations of membrane-bound whorls. In addition, phagocytic activity in the treated cells was notably increased and the level of acid lipase was elevated. These data suggest that increased levels of chondroitin sulfate can induce alterations in both metabolism and activities of RPE cells.