Effects of antiglaucoma drops on MMP and TIMP balance in conjunctival and subconjunctival tissue.

PURPOSE: To study the effects of antiglaucoma drugs on metabolism within the extracellular matrix (ECM) of the ocular surface, including corneal, conjunctival, and subconjunctival tissue. METHOD: Several antiglaucoma drugs--including beta-blockers, alpha/beta-blockers, alpha1-blocker, alpha2-agonist, and prostaglandin derivative-were topically administrated to rat eyes daily for 2 weeks or were incubated with human corneal cells or human fibroblasts for 72 hours. Thereafter, expression and enzymatic activity of the matrix metalloproteinases (MMPs), a group of enzymes proteolyzing ECM and their inhibitors, called tissue inhibitors of metalloproteinase (TIMPs), were evaluated. RESULTS: Quantitative RT-PCR revealed significantly upregulated and downregulated expression of MMPs and TIMPs, respectively, in rat conjunctival and subconjunctival tissue on the administration of alpha/beta-blockers, alpha1-blocker, alpha2-agonist, and prostaglandin derivative, suggesting that these drugs may enhance ECM degradation. However, in contrast, beta-blocker administration caused reverse effects--that is, upregulation and downregulation of TIMPs and MMPs, respectively. Enzymatic activity of MMPs in rat conjunctival and subconjunctival tissue analyzed by biochemical assay and zymography was markedly enhanced on the administration of alpha/beta-blockers, alpha1-blocker, alpha2-agonist, and prostaglandin derivative, but not of beta-blockers. Similar effects of these antiglaucoma drugs were observed in cultured human corneal cells and human fibroblast cells. CONCLUSIONS: The present experimental observations suggest that some alpha/beta-blockers, alpha1-blocker, alpha2-agonist, and prostaglandin derivative stimulate ECM degradation of ocular surface tissue by modulating the balance between MMPs and TIMPs.

Prolonged rhodopsin phosphorylation in light-induced retinal degeneration in rat models.

PURPOSE: The effects of various light-induced stresses on the retina were examined in the retinal degenerative rat model. METHODS: Retinal morphology and electroretinograms (ERGs) were analyzed after application of light-induced stress of several intensities (650, 1300, 2500, or 5000 lux). For evaluation of rhodopsin (Rho) function, the kinetics of Rho regeneration and dephosphorylation were studied by spectrophotometric analysis and immunofluorescence labeling with antibodies specifically directed toward the phosphorylated residues (334)Ser and (338)Ser in the C terminus of Rho. Retinal cGMP concentration was determined by ELISA. Expression levels of neurotrophic factors (FGF2, brain-derived neurotrophic factor [BDNF], platelet-derived growth factor [PDGF], and ciliary neurotrophic factor [CNTF]) were evaluated quantitatively by RT-PCR. RESULTS: Light intensity-dependent deterioration of ERG responses and thinning of the retinal outer nuclear layer were observed in wild-type and Royal College of Surgeons (RCS) rat retinas. Under dark adaptation after light-induced stress, the kinetics of Rho regeneration were not different between wild-type and RCS rat retinas. Rho dephosphorylation at (334)Ser and (338)Ser was extremely delayed in RCS rat retinas compared with wild-type without light-induced stress, but Rho dephosphorylation at those sites became slower in both RCS and wild-type rat retinas. In terms of expression of neurotrophic factors, almost no significant changes were observed between the animals after light-induced stress. CONCLUSIONS: The present study indicates that light-induced stress causes intensity-dependent deterioration in retinal function and morphology in wild-type and RCS rat retinas. Disruption of the phototransduction cascade resulting from slower kinetics of Rho dephosphorylation appears to be involved in retinal degeneration.

Study of effects of antiglaucoma eye drops on N-methyl-D-aspartate-induced retinal damage.

PURPOSE: To study the effects of antiglaucoma eye drops on N-methyl-D-aspartate (NMDA)-induced retinal damage. METHODS: Several antiglaucoma eye drops, beta-blockers, alpha/beta-blockers, an alpha1-blocker, an alpha2-agonist, and a prostaglandin derivative, were topically administrated to NMDA-treated rat eyes daily for 2 weeks, and the retinal thickness, the number of retrograde-labeled retinal ganglion cells (RGCs), and the results of a cDNA microarray analysis were studied. RESULTS: Intravitreal administration of NMDA caused a significant decrease in the thickness of the retinal layers and induced upregulation of glial fibrillary acidic protein (GFAP). Topical administration of beta-blockers (timolol, betaxolol, and carteolol) and a prostaglandin derivative (latanoprost) showed almost no significant effects on retinal thickness, the number of RGCs, or expression of GFAP. In contrast, the alpha/beta-blockers (nipradilol and levobunolol), the alpha1-blocker (bunazosin HCl), and the alpha2-agonist (brimonidine) showed preservation effects on retinal thickness and the number of RGCs, and marked suppression of NMDA-induced upregulation of GFAP. Among 1101 genes related to cellular regulatory mechanisms, the expression of two genes, both for insulin-like growth factors, (IGF-1) and ErbB3, was altered upon administration of the alpha/beta-blockers, the alpha1-blocker, and the alpha2-agonist. CONCLUSION: Our present study suggests that modulations of the alpha-adrenergic receptor, alpha1-blocking and alpha2-stimulation, by antiglaucoma eye drops may cause beneficial effects on NMDA-induced retinal damage in the rat.

Prolonged survival of the phosphorylated form of rhodopsin during dark adaptation of Royal College Surgeons rat.

To study rhodopsin (Rho) phosphorylation and dephosphorylation in Royal College of Surgeons (RCS) rat retina, specific antibodies toward major Rho phosphorylation sites in vivo, 334Ser or 338Ser, were prepared by immunization of authentic phosphorylated peptides in rabbit. Enzyme-linked immunosorbent assay identified that the raised antibodies exclusively recognized either the phosphorylated 334Ser or 338Ser site. In immunofluorescence labeling, both antibodies recognized photoreceptor outer segments in light-adapted retinas from Sprague-Dawley (SD), Brown-Norway (BN) and RCS rat. During dark adaptation, immunoreactivities toward phosphorylated 338Ser and 334Ser sites were diminished within several hours (0.2-2 h) in SD and BN rat retinas. However, those toward phosphorylated 338Ser and 334Ser sites were diminished within 4 to 7 days in RCS rat retinas. In vitro studies demonstrated decreased levels of both Rho phosphorylation and dephosphorylation reactions in RCS retinas. However, the dephosphorylation reaction was much more greatly affected than the phosphorylation reaction. Extremely prolonged survival of phosphorylated forms of Rho may contribute to persistent misregulation of phototransduction processes in retinal degeneration in RCS rat.

Clinical and immunologic aspects of cancer-associated retinopathy.

PURPOSE: To report clinical and immunologic aspects of cancer-associated retinopathy (CAR). DESIGN: Observational consecutive case series. METHODS: A retrospective review was made of 18 consecutive patients with cancer-associated retinopathy who had antiretinal antibody determination by Western blot testing. RESULTS: Clinically, a variety of ophthalmic observations including electroretinography impairment, retinal vessel narrowing, deterioration of visual acuity, visual field changes, and uveitis were frequently observed. As retinal autoantigens in the 18 cases, recoverin was found in all 18 cases (100%), heat shock cognate protein 70 (HSC70) was found in six cases (33%), and other proteins were found in four cases (20%). These antibodies were detected in only 60% of the patients at the initial examination, however, and then became increasingly apparent on the subsequent testing that was performed three times on serum samples obtained sequentially during the following months. CONCLUSION: For diagnosis of cancer-associated retinopathy, the presence of serum autoantibody toward recoverin is essentially required in addition to the characteristic clinical aspects noted above.

Low expression of rhodopsin kinase in pineal gland in Royal College of Surgeons rat.

The Royal College of Surgeons (RCS) rat has been extensively characterized as a model for inherited retinal dystrophy such as retinitis pigmentosa. We have found that significantly low levels of expression of rhodopsin kinase (RK) and alphaA-crystallin may be involved in the pathogenesis of retinal degeneration in the RCS rat (Invest Ophthalmol Vis Sci. 1999,40:2788-2794). In the present study, we examined the expression of photoreceptor specific proteins in the pineal gland (PG) including rhodopsin kinase (RK), arrestin and recoverin, which are known to be commonly present in both photoreceptor and PG, in order to elucidate the pathological relationship between retina and PG during retinal degeneration. Among these proteins, RK expression was significantly decreased with advancing age (3-5 weeks old) in RCS rat. However, in contrast, arrestin expression in RCS PG was comparable with control PG and no expressions of recoverin and other G-protein coupled receptor kinases (GRKs 2, 5 and 6) were detected in RCS PG during 3-5 weeks of age. By administration of nilvadipine, an effective Ca2+ antagonist that was shown to preserve RCS retinal degeneration, RK expression was significantly enhanced.

Effects of matrix metalloproteinase-3 gene transfer by electroporation in glaucoma filter surgery.

To develop gene therapy that can be applied to glaucoma-filtering surgery, we studied effects of transfection of matrix metalloproteinase-3 (MMP-3) cDNA into rabbit conjunctiva by electroporation (EP) on changes of intraocular pressure (IOP) and bleb formation after glaucoma filtering surgery. pTracer-CMV2 vector containing MMP-3 cDNA was transfected into rabbit conjunctiva by EP and MMP-3 expression was studied by reverse transcription (RT)-PCR, zymography and western blot analysis. Three days after the EP transfection of MMP-3 cDNA or vector alone into rabbit conjunctiva, trabeculectomy was performed at the place of transfection in the presence or absence of 0.04% mitomycin C (MMC). Then changes in IOPs and bleb formation were compared with each other. Expression of MMP-3 was detected in conjunctiva until 30 days after transfection by EP. Trabeculectomy following MMP-3 transfection caused significantly longer survival of filtering bleb and decreased levels of IOP in comparison with controls (trabeculectomy alone or trabeculectomy following vector transfection), and these levels were almost identical to those of trabeculectomy with MMC. The present study indicates that EP is effective to transfect some genes that promote the filtering bleb formation in glaucoma surgery, such as MMP-3 gene, and this may be potentially applicable to glaucoma-filtering surgery in glaucoma patients.

Study of pharmacological effects of nilvadipine on RCS rat retinal degeneration by microarray analysis.

In our recent study, we found that the Ca(2+) antagonist, nilvadipine caused significant preservation of photoreceptor cells in The Royal College of Surgeons (RCS) rats [Invest. Ophthalmol. Vis. Sci. 43 (2002) 919]. Here, to elucidate the mechanisms of nilvadipine-induced effects we analyzed altered gene expression of 1101 genes commonly expressed in rodent by DNA microarray analysis in the retinas of nilvadipine-treated and untreated RCS rats and SD rat. In the total number of genes, the expression of 30 genes was altered upon administration of nilvadipine to RCS rats, including several genes related to the apoptotic pathway and other mechanisms. Remarkably, neurotrophic factors, FGF-2 and Arc, known to suppress the apoptosis in the central nervous system, were up-regulated. These changes were also confirmed by real-time quantitative (Taqman) RT-PCR and Western blot analysis. Therefore, our present data suggested that administration of nilvadipine to RCS rats increases the expression of endogenous FGF-2 and Arc in retina, and potentially has a protective effect against retinal degeneration.

Role of Cys41 in the N-terminal domain of lumican in ex vivo collagen fibrillogenesis by cultured corneal stromal cells.

The keratan sulphate proteoglycan lumican regulates collagen fibrillogenesis to maintain the integrity and function of connective tissues such as cornea. We examined the role of a highly conserved cysteine-containing domain proximal to the N-terminus of lumican in collagen fibrillogenesis using site-specific mutagenesis to prepare plasmid DNA encoding wild-type murine lumican (Cys(37)-Xaa(3)-Cys(41)-Xaa-Cys-Xaa(9)-Cys) and a Cys-->Ser (C/S) mutant (Cys(37)-Xaa(3)-Ser(41)-Xaa-Cys-Xaa(9)-Cys). cDNAs were cloned into the pSecTag2A vector, and cultures of MK/T-1 cells (an immortalized cell line from mouse keratocytes) were transfected with the cDNAs. Stable transformants were selected and cloned in the presence of Zeocin. All stable transformants maintained a dendritic morphology and growth rate similar to those of parental MK/T-1 cells. Western blot analysis with anti-lumican antibody detected a 42 kDa lumican protein secreted into the culture medium of both wild-type and C/S mutant lumican cell lines. Ultrastructural analyses by transmission electron microscopy showed both cell lines to form a multi-layered stroma ex vivo, but the matrix assembled by the two cell lines differed. Compared with the mutant cell line, the wild-type cells assembled a more organized matrix with regions containing orthogonal collagen fibrils. In addition, the fibrils in the extracellular matrix formed by the mutant cell line exhibited alterations in fibril packing and structure. Immunostaining analysed by confocal microscopy showed a further difference in this matrix, with the marked occurrence of lumican and collagen I co-localization in the lumican wild-type cells, but a lack thereof in the lumican C/S mutant cells. The results indicate that the cysteine-rich domain of lumican is important in collagen fibrillogenesis and stromal matrix assembly.

Study of drug effects of calcium channel blockers on retinal degeneration of rd mouse.

In the present study, we studied drug effects of Ca(2+) antagonists on the retinal degeneration of rd mouse to evaluate their efficacy. Several kinds of Ca(2+) antagonists, diltiazem, nicardipine, nilvadipine or nifedipine were administrated intraperitoneally and thereafter retinal morphology and functions were analyzed. In addition, we performed DNA microarray analysis both in nilvadipine treated and control retinas to understand their drug effects at molecular levels. We found that nilvadipine caused significant preservation of retinal thickness in rd mouse during the initial stage of the retinal degeneration, and nicardipine showed also significant but lesser preservation than nilvadipine. However, we recognized no preservation effects of diltiazem and nifedipine. In the total 3774 genes, the expressions of 27 genes were altered upon administration of nilvadipine, including several genes related to the apoptotic pathway, neuro-survival factor, Ca(2+) metabolisms, and other mechanisms. It is suggested that some types of Ca(2+) channel blockers, such as nilvadipine and nicardipine, are able to preserve photoreceptor cells in rd mouse and can potentially be used to treat some RP patients.