Masaru Inatani

Transforming growth factor-β2 levels in aqueous humor of glaucomatous eyes

Abstract. Purpose: To determine whether clinical characteristics are correlated with increased levels of transforming growth factor-β2 (TGF-β2) in aqueous humor in glaucomatous eyes. Methods: Aqueous humor samples were collected from 91 glaucomatous eyes. Included were samples from primary open-angle glaucoma (POAG) in 40 eyes, (pseudo)exfoliation syndrome (EXS) in 18 eyes, primary angle-closure glaucoma (PACG) in 26 eyes and uveitis-related secondary glaucoma (SG) in 7 eyes. TGF-β2 in aqueous humor was assessed with a specific-capture ELISA. Results: The mean concentration (± standard error) of mature (biologically active) TGF-β2 in the aqueous humor of eyes with POAG was 293.6±33.6xa0pg/ml, significantly higher than that in eyes with PACG, EXS and SG: 147.5±28.1, 135.8±30.2 and 41.0±10.7xa0pg/ml, respectively (P=0.0006, P=0.0010 and P=0.0003; analysis of variance). The mean concentration (± standard error) of total TGF-β2 in the aqueous humor of eyes with POAG was 1647.6±124.5xa0pg/ml, not significantly different from that in eyes with PACG, EXS and SG: 1482.9±148.2, 1442.7±187.8 and 1929.0±367.6xa0pg/ml, respectively. A multivariate analysis using logistic regression showed significant correlations between mature TGF-β2 concentration and history of cataract surgery (P=0.0225) and the use of carbonic anhydrase inhibitors (P=0.0143). Conclusions: Our results indicate that increased levels of TGF-β2 may play an important role in the pathogenesis of POAG.

Growth factors and their receptors in the retina and pigment epithelium

Abstract Growth factors are regarded as factors to induce (or in some cases inhibit) growth of cells/tissues in vitro and/or in vivo . Molecules regarded as growth factors consist of six groups: the transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and insulin-like growth factor (IGF). In an attempt to introduce clinical implications of such factors in ocular diseases, in this review article, we describe the expression of growth factors and their receptors in the neural retina and retinal pigment epithelium (RPE). Also, the expression, clinical implications and therapeutic potential influence of such factors in a number of ocular diseases, such as proliferative vitreoretinopathy (PVR), epiretinal membranes, macular holes, diabetic retinopathy and retinal degeneration, are discussed. In summary, TGF-β is expressed in RPE cells under a variety of conditions, and is thought to enhance various processes in the pathogenesis of PVR in several ways such as stimulating cell-mediated gel contraction, modifying mitogenic effects of other growth factors and enhancing extracellular matrix production and the resultant fibrosis reaction. In part because of these diverse effects, TGF-β is a good candidate for adjunct use with vitrectomy for the treatment of macular holes. PDGF is another growth factor that is thought to be involved in the onset of proliferative intraocular diseases such as epiretinal membranes and PVR. PDGF is a potent mitogenic and chemotactic factor for retina-derived cells. With respect to proliferative diabetic retinopathy in particular, recent developments in clinical and basic research on the angiogenic effects of VEGF, which is also a member of PDGF family, have drawn much attention from investigators. So-called eye- and retina-derived growth factors have been shown to be identical to FGF. In both retina and RPE cells, FGF is known to induce a variety of changes in cellular proliferation, differentiation and in vivo angiogenesis. In addition to these changes, FGF is a promising neuroprotective drug against some retinal degenerative diseases. There is currently limited information on the relationship of differentiation of retinal precursor cells in the developing retina and EGF/TFG-α. Further studies on its physiological and pathological significance in the retina and RPE are required. IGF and insulin also are thought to play important roles in the development of diabetic retinopathy. Recent insight into the effects of VEGF, in addition to those of IGF/insulin, has modified our thinking of contribution of this growth factor to the proliferative and angiogenic response of the retina in diabetes. Taken together, our knowledge of the effects of growth factors on the eye has advanced dramatically because of the recent advances in molecular biology and cell biology. A number of investigators around the world are currently performing intensive research in an attempt to understand the significance of these various factors in the pathogenesis of ocular diseases. It is reasonable to assume that novel concepts in the treatment of many refractory ocular diseases will result from such studies.

Neuroprotective effects of brain-derived neurotrophic factor in eyes with NMDA-induced neuronal death

PURPOSEnTo determine if brain-derived neurotrophic factor (BDNF) has a neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced cell death in retina.nnnMETHODSnNMDA was injected into the vitreous of rat eyes. NMDA-induced neuronal death was measured by morphometric analyses on cell counts of ganglion cell layer cells and thickness of retinal layers. Also, we conducted additional experiment using retrograde labeling with a fluorescent tracer (Fluoro-Gold) for exact counting of retinal ganglion cells (RGCs). In addition, intravitreal glutamate levels were measured with the use of a high-performance liquid chromatography (HPLC) system.nnnRESULTSnMorphometric analysis of retinal damage in NMDA-injected eyes showed that BDNF could protect inner retinal cells from glutamate receptor-mediated neuronal death. Also, counts of RGCs labeled with a fluorescent tracer showed that BDNF could protect RGCs from glutamate receptor-mediated neuronal death. Furthermore, measurements of intravitreal glutamate levels indicated an increase in this excitatory amino acid in the vitreous after NMDA injection.nnnCONCLUSIONSnExogenous BDNF can protect inner retinal cells (possible RGCs and amacrine cells) from NMDA-induced neuronal death. However, increased intravitreal glutamate levels in response to NMDA-mediated neurotoxicity may augment retinal degeneration.

Proteoglycans in retina.

In this article, we summarize the roles of proteoglycans in retinal tissue. Chondroitin sulfate and heparan sulfate proteoglycans are the major constituents in proteoglycans expressed in retinal tissue. Soluble heparan sulfate proteoglycans are found in the extracellular matrices of the basement membrane, such as the inner limiting membrane and Bruchs membrane, whereas heparan sulfate proteoglycans with their membrane-binding domain are localized primarily in the neurites of retinal neuronal cells, indicating their role as receptors for cytokines. The distribution of chondroitin sulfate proteoglycans is classified into two regions: nerve fiber-rich layers such as the optic nerve, inner plexiform layer and outer plexiform layer, and the interphotoreceptor matrix (IPM). The expression in the nerve fiber-rich layers of several chondroitin sulfate proteoglycans, such as neurocan and phosphacan, is restricted in the nervous tissues, and is upregulated as retinal development proceeds, then decreases after maturation of the retina. In vitro data suggest that these proteoglycans regulate axon guidance and synapse formation during the development of nervous tissue. In contrast, in adult vertebrate retina, the IPM is a rich source of chondroitin sulfate proteoglycans. Histologic data from animals with experimental retinitis pigmentosa, and the existence of the hyaluronan-binding domain in their core proteins, indicate that these proteoglycans contribute to the structural link between the neural retina and retinal pigment epithelium via the interaction with hyaluronan, which is also abundant in the IPM. Furthermore, several chondroitin sulfate proteoglycans in the nerve fiber-rich layers contain the hyaluronan-binding domain, so it is likely that the interaction of proteoglycans with hyaluronan plays an important role in neural network formation in the central nervous system.

Proteoglycans in the eye.

Purpose Various proteoglycans are expressed in ocular tissues. We investigated and reviewed the distribution and the potential roles of proteoglycans in cornea, trabecular meshwork, and retinal tissues. Methods Immunohistochemical studies were performed in rat ocular tissues. The concentration of transforming growth factor (TGF)-β2, which regulates the expression of proteoglycans in aqueous humor from human glaucomatous eyes, was evaluated by enzyme-linked immunosorbent assay (ELISA). In retinal tissues, we examined the localization of 2 soluble nervous tissue-specific chondroitin sulfate proteoglycans, neurocan and phosphacan, by immunohistochemical analysis, then investigated the effect on the neurite outgrowth of cultivated retinal ganglion cells. Results The expression of chondroitin sulfate in stroma was upregulated at early postnatal stages and reduced during development in rat eyes. In trabecular meshwork tissues, immunohistochemical studies showed the intense expression of decorin. Moreover, elevated levels of TGF-β2 in the aqueous humor from glaucomatous patients were observed. In retinal tissues, neurocan and phosphacan were expressed mainly in nerve fiber-rich layers during rat postnatal stages. In vitro, the neurite extension from retinal ganglion cells was inhibited by neurocan and phosphacan. Conclusions Soluble extracellular proteoglycans in corneal and trabecular meshwork tissues contribute to the stromal transparency in the corneal tissues and the resistance of the aqueous humor outflow in trabecular meshwork tissues. In retinal tissues, chondroitin sulfate and heparan sulfate proteoglycans are not only secreted into the extracellular space of retinal tissues but also expressed in the membrane of the retinal cells, contributing to the neural network formation and the maintenance of the interphotoreceptor matrix.

Novel Mutations in the Myocilin Gene in Japanese Glaucoma Patients

Myocilin is a gene responsible for juvenile onset primary open angle glaucoma (POAG) mapped as the GLC1A locus and, many mutations have been reported worldwide. Some mutations were found not only in patients with juvenile onset POAG, but also in patients with late onset POAG and in patients with normal tension glaucoma. To investigate the mutation prevalence in Japan, we performed a mutation analysis in 140 unrelated Japanese patients. We have identified the 10 sequence variants, of which four were highly probable for disease‐causing mutations (Arg46ter, Arg158Gln, Ile360Asn, and Ala363Thr), and six polymorphisms (Gln19His, Arg76Lys, Asp208Glu, Val439Val, Arg470His, and Ala488Ala). Thus, myocilin mutations were found at the rate of 4/140 (2.9%) probands, similar to previous reports with other ethnic populations. Hum Mutat 16:270, 2000.

Phacoemulsification, intraocular lens implantation, and trabeculotomy to treat pseudoexfoliation syndrome

Purposes: To determine the long‐term risk/benefit ratio of phacoemulsification and intraocular lens (IOL) implantation combined with trabeculotomy to manage eyes with pseudoexfoliation syndrome and co‐existing cataract. Setting: Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine; Kurihara Eye Clinic; Departments of Ophthalmology, Tenri Hospital, Kumamoto University, and Matsue Red Hospital; Nagata Eye Clinic, Japan. Methods: This multicenter retrospective study comprised 49 eyes of 36 patients with pseudoexfoliation syndrome and co‐existing cataract who had the combined procedure for uncontrolled intraocular pressure (IOP) (>21 mm Hg) even on antiglaucoma medication. Results: After a mean follow‐up of 20.0 months ± 13.2 (SD), IOP in all 49 eyes was well controlled (≤21 mm Hg). Mean IOP at the final examination was 14.6 ± 2.6 mm Hg on a mean of 0.9 ± 0.8 glaucoma medications. Complications included an IOP spike in 11 eyes and fibrin exudation in 1 eye. Conclusion: Phacoemulsification and IOL implantation combined with trabeculotomy was an effective treatment for patients with pseudoexfoliation syndrome and cataract.

Pharmacokinetics and pharmacodynamics of acetazolamide in patients with transient intraocular pressure elevation

AbstractObjective: To characterize the pharmacokinetics and pharmacodynamics of acetazolamide in patients with transient intraocular pressure (IOP) elevation and to provide individual patients with the optimal dosage regimen for this drug.nn Methods: We studied 17 patients with transient IOP elevation, who were given 62.5–500u2009mg acetazolamide orally as single or repetitive doses. Plasma acetazolamide concentration and IOP were measured at approximately 1, 3, 5, and 9u2009h after the last acetazolamide administration. Pharmacokinetics and pharmacodynamics were analyzed by nonlinear mixed-effect modeling using the program NONMEM.nn Results: The plasma concentration profile of acetazolamide was characterized by a one-compartment model with first-order absorption. The apparent oral clearance was related to the creatine clearance (CCR) which was estimated by the Cockcroft and Gault equation, as follows: 0.0468u200a·u200aCCRu200alu200a·u200ah−1. The estimated apparent oral volume of distribution, first-order absorption rate constant, and absorption lag time were 0.231u2009lu200a·u200akg−1, 0.821u200a·u200ah−1, and 0.497u2009h, respectively. IOP after oral acetazolamide administration was characterized by an Emax model. The maximal effect in lowering the IOP (Emax) was 7.2u2009mmHg, and the concentration corresponding to 50% of the maximal effect (EC50) was 1.64u2009μgu200a·u200aml−1. As 70% of Emax was achieved at a plasma concentration of 4u2009μgu200a·u200aml−1, this concentration was considered satisfactory for lowering IOP. The recommended dosage was calculated so that the minimum plasma concentration at steady state exceeded this target concentration; 250u2009mg t.i.d., 125u2009mg t.i.d., 125u2009mg b.i.d., and 125u2009mg once daily for the patients with CCR values of 70, 50, 30, and 10u2009mlu200a·u200amin−1, respectively.nn Conclusion: Measuring plasma concentrations of acetazolamide and subsequent pharmacokinetic and pharmacodynamic analyses are useful for estimating its concentration-dependent effectiveness in lowering the IOP in individual patients. The dosage regimen presented in this study is expected to improve the benefits of acetazolamide pharmacotherapy in most elderly patients with transient rises in IOP following intraocular surgery.

Trabeculotomy combined with phacoemulsification and implantation of an intraocular lens for the treatment of primary open-angle glaucoma and coexisting cataract

BACKGROUND AND OBJECTIVEnThe authors previously reported the usefulness of trabeculotomy ab externo for the treatment of primary open-angle glaucoma in adult patients. In an attempt to elucidate the long-term risk-to-benefit ratio of this surgical modality in combination with cataract surgery, the authors conducted a retrospective study of the surgical effects and complications of a triple procedure: phacoemulsification, implantation (of an intraocular lens), and trabeculotomy (PIT).nnnPATIENTS AND METHODSnThe authors conducted a retrospective study of patients treated with PIT at multiple hospitals. Intraocular pressure (IOP) and visual function data were obtained from patients after PIT as an initial surgical treatment in cases where antiglaucoma medications failed to resolve uncontrolled IOP (higher than 21 mm Hg). Included in this study were 96 eyes of 64 patients with primary open-angle glaucoma and coexisting cataract. The mean follow-up period was 22.6 +/- 14.7 months (range 3-56 months).nnnRESULTSnIn 94 (98%) of the 96 eyes, the IOP was well controlled, having achieved a level of 21 mm Hg or lower at the final examinations. The mean preoperative IOP of the 33 eyes that underwent the triple procedure using a single flap method (PIT-I) was 24.3 +/- 3.9 mm Hg, with an average of 2.1 +/- 1.1 medications. At the final examinations, the mean IOP had dropped to 16.0 +/- 1.2 mm Hg, with an average of 1.2 +/- 1.2 medications. The mean preoperative IOP of the 63 eyes that underwent the triple procedure using a double flap method (PIT-II) was 26.2 +/- 6.2 mm Hg, with an average of 1.9 +/- 1.2 medications. At the final examination, the mean IOP for this group was 15.6 +/- 2.9 mm Hg, with an average of 1.0 +/- 0.9 medications.nnnCONCLUSIONnThe long-term results from this multicenter study showed that the triple procedure, PIT, can be useful and effective as an initial surgical treatment for open-angle glaucoma in glaucoma patients with coexisting cataract.

Dual effects of interleukin-1β on N-methyl-d-aspartate-induced retinal neuronal death in rat eyes

In this study we determine if interleukin-1beta (IL-1beta) modulates N-methyl-D-aspartate (NMDA)-induced retinal damage. Sprague-Dawley rats were anesthetized with inhalation of halothane, after which a single injection of 5 microl of IL-1beta (0.1 to 10 ng/eye) (and/or IL-1 receptor antagonist (IL-1ra)) for experimental eyes was administered. Two days later (or simultaneously), NMDA (20 nmol) was injected into the vitreous space. One week later, each eye was enucleated and transverse sections were subjected to morphometric analysis. Enzyme-linked immunosorbent assay (ELISA) was conducted for the determination of IL-1beta levels in retina. Immunohistochemical and immunoblot studies were also performed. In eyes that received an intravitreal injection of IL-1beta (0.1 to 10 ng/eye), significant thinning of the inner plexiform layer (IPL) was observed (P<0.05). Immunohistochemical and ELISA studies demonstrated upregulated expression of IL-1beta in retinas that had undergone NMDA injection. Treatment with 10 ng of IL-1ra induced a protective effect against NMDA-induced retinal damage. Pretreatment with IL-1beta induced a significant protective effect on NMDA-induced retinal damage. Our studies suggest that IL-1beta induces neuronal cell death directly, as shown by the protective effects of IL-1ra, but has a protective effect on NMDA-induced retinal damage indirectly after an incubation time of at least 2 days.