Yoko Iizuka

Matrix metalloproteinases in human diabetic and nondiabetic vitreous.

Purpose To compare matrix metalloproteinase (MMP) activities in human vitreous samples from patients with diabetic retinopathy (DR) and other vitreoretinal diseases, and to investigate the factors influencing the MMP activities in human DR vitreous samples. Methods Thirty-one diabetic and 17 nondiabetic vitreous samples (from nine patients with macular holes and eight patients with epiretinal membranes) were examined. Samples collected at the time of pars plana vitrectomy were subjected to substrate zymography to conduct a quantitative analysis of MMP activity. Immunoblotting against antihuman MMP-1, 2, and 9 was performed to identify MMP in vitreous samples. The effects of posterior vitreous detachment (PVD), vitreous hemorrhage, proliferative membrane, traction detachment, and cystoid macular edema on MMP activities were investigated. Results All vitreous samples from both DR and non-DR patients showed a single band at the position of 72 kD, corresponding to MMP-2. Another band at 99 kD, corresponding to MMP-9, was detected significantly more often in DR samples than in non-DR samples: 45.2% and 0%, respectively (P = 0.0007). The number of samples showing a band from MMP-9 was significantly higher in partial PVD samples than in complete PVD samples: 66.7% and 15.4%, respectively (P = 0.001). Conclusion The results indicated that MMP-9 may be involved in DR and that partial PVD may be related to the MMP-9 activity in DR.

Differences in nitric oxide production: a comparison of retinal ganglion cells and retinal glial cells cultured under hypoxic conditions

The aim of this study was to compare the effects of hypoxia on nitric oxide synthase (NOS) expression and the production of NO between isolated retinal ganglion cells (RGCs) and retinal glial cells. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the presence of neuronal NOS mRNA, inducible NOS mRNA, and endothelial NOS mRNAs in the two cell types. RGCs and retinal glial cells were separately cultured under hypoxic (10% O(2)) or control (20% O(2)) conditions. Changes in NOS-mRNA expression were quantified by real-time PCR, and nitrite in the medium was measured up to 96 h of culture. The effects of non-NOS- and iNOS-selective inhibitors on hypoxia-induced release of nitrite in the culture medium were evaluated. RT-PCR revealed the presence of three types of NOSs in the two types of cultured cells. Hypoxic culture conditions significantly changed the expression of all NOS mRNAs in retinal glial cells but not in RGCs. NO production showed significant changes corresponding to those of NOS mRNAs in retinal glial cells but not in RGCs, and both NOS inhibitors significantly reduced hypoxia-induced nitrite release in retinal glial cells. Retinal glial cells but not RGCs may be the major source of NO under hypoxic conditions.

Neuroprotective effects of nipradilol on purified cultured retinal ganglion cells.

PurposeTo investigate effects of nipradilol, a nonselective &bgr;- andbgr;-and selective &agr;1-receptor antagonist and a potential nitric oxide releaser, on retinal ganglion cells purified and cultured in a serum-free medium. MethodsRetinal ganglion cells were isolated from 2-day-old Sprague-Dawley rats by means of two-step panning. A series of nipradilol (10−5, 10−6, 10−7, 10−8, 10−9, and 10−10-mol/L) or vehicle solutions were administered to the culture medium for 48 hours, and the survival rate of retinal ganglion cells was evaluated using a newly developed system that evaluates the survival rate in small and large retinal ganglion cells separately. The effects of timolol maleate or bunazosin (10−5, 10−6, 10−7, and 10−8-mol/L) solutions on retinal ganglion cells survival were also evaluated. The survival rate was evaluated after 10−5-mol/L c-PTIO (2-[4-carboxyphenyl]-4,4,5,5 tetramethylimidazoline-1-oxyl-3-oxide potassium salt), a nitric oxide scavenger, was administered to retinal ganglion cells with 10−5-mol/L nipradilol. ResultsNipradilol significantly increased the survival rate of both small and large retinal ganglion cells in a concentration-dependent manner compared with the controls. The maximum survival rate improvement of small and large retinal ganglion cells was 29.1% and 14.5%, respectively. Although timolol maleate and bunazosin did not affect the survival rate, 10−5-mol/L c-PTIO significantly inhibited the nipradilol-induced survival rate improvement by 69.9% in small retinal ganglion cells and by 91.6% in large retinal ganglion cells. ConclusionNipradilol improves the survival rate of cultured postnatal rat retinal ganglion cells, and the nitric oxide generated from nipradilol may contribute to this effect.

Isopropyl unoprostone increases the activities of matrix metalloproteinases in cultured monkey ciliary muscle cells.

PurposeThe mechanism by which the prostaglandin F 2&agr; -related antiglaucoma compound isopropyl unoprostone (referred to as unoprostone) reduces intraocular pressure is largely unknown. Another prostaglandin F 2&agr; -related compound, latanoprost, influences the activities of matrix metalloproteinases in ciliary muscle. Unoprostone ophthalmic solution is metabolized to oxidized metabolites, mainly M1 and M2, in the eye. The aim of this study was to investigate whether intraocular metabolites of unoprostone, M1 and M2, change the metalloproteinase activity in cultured monkey ciliary muscle cells. Materials and MethodsMonkey ciliary muscle cells and trabecular meshwork cells were grown separately to confluence in monolayer cell cultures. M1 (10 nM, 100 nM, or 1 &mgr;M), M2 (10 nM, 100 nM, or 1 &mgr;M), 100 nM prostaglandin F 2&agr;, or vehicle solutions were added to each culture medium for 48 hours. The media were then assayed to measure metalloproteinase activities quantitatively by means of substrate zymography. ResultsCompared with the vehicle controls, M1, M2, and prostaglandin F 2&agr; significantly increased the metalloproteinase-2 activity in cultured ciliary muscle cells in a dose-dependent manner, but did not affect the metalloproteinase-2 activity in cultured trabecular meshwork cells. All experimented prostaglandins slightly increased metalloproteinase-9 activity in ciliary muscle cells, although these changes were not significant. ConclusionsThe current results show that unoprostone influences the metabolism of the extracellular matrix in the ciliary muscle and that remodeling of the extracellular matrix in the ciliary muscle may be a possible mechanism by which unoprostone ophthalmic solution reduces intraocular pressure.

Effects of Isopropyl Unoprostone Ophthalmic Solution on Cultured Rabbit Corneal Epithelial Cells

Purpose: To investigate the effects of isopropyl unoprostone (referred to as unoprostone) ophthalmic solution on the barrier function of cultured rabbit corneal epithelium grown on permeable supports. Methods: Rabbit corneal epithelial cells cultured on collagen-coated filter inserts were administered one of the following for 30 min: unoprostone in vehicle solution (polysorbate 80), unoprostone in vehicle solution with a preservative (benzalkonium chloride), preservative only or vehicle only. For a control, no chemicals were added to the medium. After administration, the transepithelial electrical resistance (TER) measurement, a sensitive method by which to investigate the barrier function, and morphological observation using phase-contrast microscopy were performed before exposure and at 0.5, 1, 3, 6, 12, 24, 48 and 72 h after exposure. The transmission electron-microscopic observation was performed before and 72 h after exposure in all experimental conditions. Results: The cells exposed to unoprostone with the preservative showed a significant decrease in the TER, although no morphological changes were observed. The corneal epithelial cells exposed to unoprostone without preservative, the vehicle only or the preservative only did not show any differences from the control group at any measurements. Conclusion: The corneal barrier function is damaged by a combined solution of unoprostone and preservative, but not by a single solution of unoprostone, in vitro.

Dual action of nitric oxide on purely isolated retinal ganglion cells

Purpose. The role of nitric oxide (NO) in the survival of retinal ganglion cells (RGCs) was investigated. Methods. RGCs were purely isolated from postnatal Sprague-Dawley rats by 2-step panning and were cultured in chemically defined serum free medium. An NO releaser, S-nitroso-N-acetylpenicillamine (SNAP: 500 µM, 250 µM, 100 µM, 10 µM, 1 µM, 100 nM, and 10 nM), an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO: 100 µM, 33 µM, 10 µM, 1 µM), mixture of 100 µM SNAP and 33 µM c-PTIO, N(G)-nitro-L-arginine methyl ester (L-NAME: 10 mM, 5 mM, 500 µM, 100 µM or 10 µM), or their vehicles were added to the medium of pure RGC culture for 48 hr. Survival rates of small and large RGCs were determined separately by flow cytometry. Results. At =100 µM, SNAP significantly reduced RGC survival in a concentration dependent manner. At =1 µM, SNAP significantly increased survival, particularly of large RGCs. c-PTIO and L-NAME reduced the survival rates concentration-dependently. A mixture of 100 µM SNAP and 33 µM c-PTIO significantly improved RGC survival compared with when they were added on their own. Conclusions. These results indicate that NO exhibits neuroprotective and neurotoxic actions on RGCs and that low concentrations of NO may be beneficial for the survival of neonatal RGCs in vitro.