Decrease of ATP by mitochondrial m-calpain inhibitory peptide in the rat retinas.

Activations of mitochondrial calpains cause apoptosis-inducing factor-dependent apoptosis of retinal photoreceptor cells in the Royal College of Surgeons (RCS) rat, an animal model of retinitis pigmentosa. In the present study, we attempted to develop specific inhibitors of mitochondrial calpains that would prevent the retinal degeneration. We examined the inhibitory potency of 20-mer peptides of the m-calpain for mitochondrial calpains activity, determined the inhibitory regions, and conjugated the cell-penetrating peptides (CPP). The cytotoxicity and delivery of the peptide was evaluated using mouse photoreceptor-derived 661W cells. After intravitreal injection of the peptide in RCS rats, we examined the peptide delivery to the retina, photoreceptor cell death numbers, responses of the electroretinogram (ERG), concentrations of intracellular ATP, and changes of retinal morphology. Results showed that one of the peptides inhibited the activity of the mitochondrial m-calpain. The HIV-1 tat-conjugated m-calpain peptide, HIV-Nm, could preserve the inhibitory potency of the mitochondrial m-calpain, and penetrate into the 661W cells. While intravitreal injection of HIV-Nm made it possible to deliver to the retina, it did not prevent photoreceptor cell death. Furthermore, it caused the ERG attenuation and the decrease in the intracellular ATP only a day after the injection. Although HIV-Nm did not cause histological change of the retina after 1 or 2 days of the administration, the morphological abnormality of the retina was observed after 3-14 days. Our results demonstrated that HIV-Nm failed to prevent the photoreceptor cell death, but rather caused the attenuation of ERG response and the decrease of ATP.

Inhibitory effects of trehalose on malignant melanoma cell growth: implications for a novel topical anticancer agent on the ocular surface.

Purpose. To investigate the inhibitory effects of trehalose on malignant melanoma cell growth. Methods. We cultured human malignant melanoma cells in a medium containing trehalose (control/2.5%/5.0%/7.5%/10.0%) and used the MTT assay to evaluate the growth activities. Subsequently, trehalose was topically instilled on subconjunctivally inoculated melanoma cells in F334/NJcl-rmu/rmu rats, followed by a histopathological evaluation of tumor growth. Using flow cytometry, we compared the distribution of the cell cycle, rate of apoptotic cells, and intracellular factors related to the cell cycle in cultured melanoma cells after trehalose treatment. Results. The MTT study showed that proliferation of melanoma cells was significantly inhibited by ≧ 5% of trehalose concentrations in the culture media. Subconjunctivally inoculated melanoma cell masses were significantly smaller in eyes administered trehalose as compared to controls. Flow cytometry analyses demonstrated that the trehalose groups had increased rates of G2/M phase cells and apoptotic cells in the cell culture. These cells also exhibited increased expressions of cell-cycle inhibitory factors. Conclusions. The current results show trehalose inhibits malignant melanoma cell growth by inducing G2/M cell cycle arrest and apoptosis, suggesting trehalose as a potential candidate for a topical agent to inhibit proliferation of malignant tumor cells of the ocular surface.

Effects of trehalose on VEGF-stimulated angiogenesis and myofibroblast proliferation: implications for glaucoma filtration surgery.

PURPOSE: To investigate whether trehalose inhibits VEGF-stimulated or inflammatory angiogenesis and the proliferation of myofibroblasts. METHODS: Normal human dermal fibroblasts and human umbilical vein endothelial cells (HUVECs)were cocultured in trehalose-containing medium (2.5/5.0/7.5/10.0%) with or without VEGF (10 ng/mL). After 11 days, the area, length, joint, and path of neovascularization were evaluated. The effect of topical trehalose on corneal neovascularization was examined in vivo by treating Balb/c mice with alkali burn-induced corneal neovascularization. After 14 days of trehalose treatment, corneal vessels were visualized in flatmounts. The expressions of VEGFR2, phospho-VEGFR2, and vimentin were observed. Then a separate coculture model of the myofibroblasts and HUVECs was used to observe the morphologic changes of the myofibroblasts by trehalose. Furthermore, myofibroblasts were cultured with trehalose to examine the cytokeratin and E-cadherin expressions. RESULTS: In the in vitro models, there was a significant trehalose dose-dependent inhibition of neovascularization. In the in vivo alkali burn models, corneal neovascularization was significantly inhibited by treatments using ≥ 2.5% trehalose eyedrops. The expressions of VEGFR2, phospho-VEGFR2, and vimentin were downregulated by trehalose. When trehalose was added to the medium, the myofibroblasts were transformed into epithelial cell-like cells. The transformed myofibroblasts expressed cytokeratin but not E-cadherin. CONCLUSIONS: Trehalose prevents angiogenesis by partially downregulating VEGFR2 expression. In addition, trehalose inhibits the proliferation of myofibroblasts partially by inducing mesenchymal-epithelial transition. These findings suggest that trehalose has potential for use as a new agent that can control angiogenesis and fibrosis and potential for use in glaucoma surgery.

Inhibitory effects of trehalose on fibroblast proliferation and implications for ocular surgery.

Trehalose is a disaccharide which plays an important role in preserving cells from completely dehydrated circumstances. In this study, we investigated effects of trehalose on proliferative activity of fibroblasts and epithelial cells both in vitro and in vivo. As in vitro assessment, normal human dermal fibroblasts and normal human epidermal keratinocytes were cultured in media containing various concentrations of trehalose. Growth activities of cells were evaluated with MTT assay and diff-quick™ staining. Expressions of vimentin and α smooth muscle actin (α-SMA) changed by trehalose were semiquantitatively measured by Western blot. As an in vivo study, 5% or 10% trehalose was topically instilled onto rabbit eyes after simple conjunctival incision or trabeculectomy. Condition of the surgical wound was evaluated by morphologically and immunohistochemically using isolectin B4 and antibodies specific for vimentin and α-SMA. Intraocular pressures (IOPs) after trabeculectomy were compared between eyes treated with trehalose and 0.04% mitomycin C (MMC). Results obtained by in vitro experiments showed that growth activities of cultured fibroblasts and keratinocytes were inhibited by trehalose in a dose-dependent manner. Fibroblasts were strongly inhibited by trehalose concentrations ≧ 5% of trehalose, whereas keratinocytes were less inhibited compared to fibroblasts. Expressions of vimentin and α-SMA were reduced by trehalose. With in vivo experiments, postoperative application of trehalose resulted in less firm adhesion between conjunctiva and sclera compared to controls. Immunohistochemical studies showed reduced staining of isolectin B4, vimentin and α-SMA in conjunctival wounds treated by topical trehalose. Also, after trabeculectomy, IOP remained in a low range during instillation of topical trehalose solution. We concluded that trehalose has inhibitory effects on proliferation of fibroblasts and vascular tissues, partially due to inhibition of transformation of fibroblasts into myofibroblasts in wound tissues. The present results imply that trehalose can be a potential agent for preventing postoperative fibrous scar formation after ocular surgery such as glaucoma filtration surgery.