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Myocilin (MYOC) mutations are associated with juvenile- and adult-onset primary open-angle glaucoma (POAG). The purpose of this study was to determine whether MYOC gene mutations are associated with normal-tension glaucoma (NTG). The prevalence of MYOC mutations was determined in 80 Japanese NTG patients and 100 control subjects. In addition, the expression of mutant MYOC was determined by transforming COS-1 cells with five myocilin variants (R158Q, D208E, I360N, A363T, and I477S) and examining whether myocilin was present in the cultured cells and/or the culture medium by western blotting. Six different nucleotide sequence variants, R46Stop, R76K, R158Q, D208E, A488A, and one in the 3′ non-coding region, were identified in 80 NTG patients. Variants in codon 46 (R46Stop), codon 158 (R158Q), and codon 488 (A488A) were not found in the 100 normal controls. The frequency of other sequence changes (R76K, D208E, and 3′ non-coding) in NTG patients did not differ significantly from the frequencies in the control subjects. COS-1 cells transfected with the wild type, R158Q, or D208E variants secreted myocilin into the culture medium. On the other hand, the detected myocilin was significantly reduced in the medium of cells transfected with the I360N, A363T, or I477S variants that were previously identified as mutations for POAG. Definitive evidence of MYOC variants associated with NTG was not found.

The effect of nitric oxide (NO) on the contractile tone of Müller cells was investigated. Müller cells, isolated from the retina of chick embryos, were cultured on thin sheets of silicone. S-Nitro-N-acetyl-DL-penicillamine (SNAP), an NO donor, was applied in the presence or the absence of carboxyl phenyltetramethylimidazole oxide (C-PTIO), an NO scavenger. The contractile tone of Müller cells was assessed by the extent of wrinkles created on the silicone sheets. The change of contractile tone was evaluated quantitatively by digitizing the photograph before and after the application of SNAP. Relaxation of wrinkles was induced by SNAP. C-PTIO inhibited the SNAP-induced relaxation of wrinkles. These results suggest that NO affects the contractility of embryonic Müller cells and could thus modulate ocular development.

We investigated the proliferation of scleral cells in response to several growth factors in vitro to elucidate the mechanism of scleral growth in visual deprivation myopia. Scleral chondrocytes and scleral fibroblasts were cultured separately in 24-well culture dishes. The number of plated cells was counted after the addition of basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGF-alpha), TGF-beta, insulin-like growth factor I (IGF-I), IGF-II, platelet-derived growth factor AA (PDGF-AA), PDGF-AB and PDGF-BB. All the growth factors studied, except for PDGF-AA, stimulated the proliferation of both scleral chondrocytes and scleral fibroblasts. bFGF showed the highest effect. TGF-beta caused morphologic changes in both scleral chondrocytes and scleral fibroblasts. Various growth factors stimulated the proliferation of scleral chondrocytes and scleral fibroblasts in a similar manner. bFGF was a potent growth stimulator and TGF-beta was suggested to be a growth regulator on scleral chondrocytes and scleral fibroblasts.

We assayed the proliferation of porcine retinal pigment epithelial (RPE) cells, bovine melanotic and amelanotic RPE cells, and bovine aortic endothelial cells exposed to 20, 10 and 5% oxygen and compared their responses to oxygen and antioxidative enzymes (superoxide dismutase and catalase). Irrespective of the cell type, the cell growth was optimal in 10% oxygen that is most closely approximating to the oxygen concentration prevailing in the cellular environment of the choroid and the retina in vivo. However, the effects of oxygen concentrations were cell specific because bovine endothelial cells were influenced by lowering of oxygen concentrations more significantly than bovine and porcine RPE cells. Moreover, addition of antioxidative enzymes caused significant improvement in growth of porcine RPE cells, but had no significant effects on bovine RPE cells. On the contrary, the bovine vascular endothelial cells represented the only one cell type significantly inhibited by antioxidative enzymes, i.e., a decrease in reactive intermediates of oxygen was seen in the media. Our results show that responses of vascular endothelial cells to reactive species of oxygen were distinctly different from those of RPE cells and more easily influenced by the environment related to hypoxia than RPE cells.