Masayuki Takeyama

Versican and Fibrillin-1 Form a Major Hyaluronan-Binding Complex in the Ciliary Body

PURPOSE In this study, biochemistry, molecular biology, immunohistochemistry, and electron microscopy techniques were used to examine whether versican, which is known to bind fibrillin-1, interacts with fibrillin-1 in the ciliary body and vitreous, and whether the versican in this complex binds to hyaluronan. METHODS The new polyclonal antibodies against the amino and carboxyl termini of versican were raised and characterized. The mRNA expression levels of versican and fibrillin-1 were analyzed by RT-PCR and real-time PCR, and their protein levels were evaluated by Western blot analysis and immunohistochemistry. Isolation of versican bound to fibrillin-1-containing microfibrils from ciliary bodies was performed by extraction studies. Slot-blot analyses and rotary shadowing electron microscopy were applied to identify versican associated with fibrillin-1-containing microfibrils after gel filtration chromatography and density gradient centrifugation. RESULTS The newly prepared polyclonal antibodies recognized amino and carboxyl termini of chicken versican. Versican, principally V0 and V1, was found to be securely bound to fibrillin-1-containing microfibrils, forming a major hyaluronan-binding structure in the ciliary nonpigmented epithelium. In addition, Western blot analysis revealed two cleaved complexes, the carboxyl-terminal end of versican bound to fibrillin microfibrils and the amino terminal end of versican bound to hyaluronan in the vitreous body. CONCLUSIONS Fibrillin-1, versican, and hyaluronan form a unique complex in the ciliary nonpigmented epithelium, and two cleavage products of this complex were shown to exist in the vitreous body. This newly clarified fibrillin-versican-hyaluronan (FiVerHy) complex and its cleavage products may be indispensable for the physiological properties important to the ciliary body and vitreous.

Effect of Infliximab on Tumor Necrosis Factor-Alpha-Induced Alterations in Retinal Microvascular Endothelial Cells and Retinal Pigment Epithelial Cells

PURPOSE Tumor necrosis factor-alpha (TNF-α) may disrupt the extracellular matrix components comprising the blood-retinal barrier (BRB) in patients with posterior uveitis, such as Behçets disease. We investigated changes in the mRNA expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in human BRB cells in the presence of TNF-α in vitro and examined the effect of infliximab addition. METHODS Cells were cultured in the presence or absence of TNF-α, and TNF-α-exposed cells were treated with or without infliximab. We measured the expression levels of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 mRNA in human retinal microvascular endothelial ACBRI181 cells and retinal pigment epithelial ARPE-19 cells by real-time polymerase chain reaction. The cell-derived proteins degraded by MMP were observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. RESULTS Expression of MMP-3 increased and TIMP-1 decreased in the presence of 10 ng/mL TNF-α in ACBRI181 cells. Expression of MMP-1 increased and TIMP-2 decreased in the presence of 10 ng/mL TNF-α in ARPE-19 cells. These altered levels of expression were reversed by the addition of infliximab. The cell-derived proteins degraded by MMP-1 and -3 were detected in each set of cells. CONCLUSIONS The presence of TNF-α altered expression of MMPs and TIMPs in cells comprising the BRB, and infliximab counteracted this alteration.

Competitive binding of heparin with hyaluronan to a specific motif in SPACR

The critical hyaluronan binding motif (HABM) in sialoprotein associated with cones and rods (SPACR) has already been determined. As sialoproteoglycan associated with cones and rods, another interphotoreceptor matrix molecule, binds to chondroitin sulfate and heparin with or without the employment of HABMs, respectively, we evaluated and compared the binding of these glycosaminoglycans to SPACR. A western blotting study in combination with inhibition assays showed that heparin bound specifically to SPACR. A series of GST fusion proteins covering the whole SPACR molecule narrowed down the region responsible for the binding. Finally, a site‐directed mutagenesis assay demonstrated that the critical HABM also acts as a specific binding site for heparin. These results were supported with mutual inhibitions by hyaluronan and heparin in analyses using GST fusion proteins and native SPACR derived from retina. Thus, these glycosaminoglycans bind to SPACR in a different manner than to sialoproteoglycan associated with cones and rods. The competitive binding between hyaluronan and heparin to SPACR, mediated through the identical HABM, may dominate the functions of SPACR, in turn involving physiological and pathological processes involved in retinal development, aging and other related disorders.

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

BackgroundPrevious reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD) after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A) and in vitro angiogenesis in retinal pigment epithelium (RPE).ResultsWe cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium–derived factor (PEDF). We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased.ConclusionsRPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

Increase in matrix metalloproteinase-2 level in the chicken retina after laser photocoagulation.

We investigated the levels of matrix metalloproteinase‐2 (MMP‐2), which has been implicated in various vitreoretinal diseases, in the retina after laser photocoagulation (LPC).

Characterization of a motif for specific binding to hyaluronan in chicken SPACR

The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA‐binding motif found near the carboxyl‐terminus of SPACR was mutated by site‐directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA‐affinity column under both reducing and non‐reducing conditions. These results revealed that the specific putative HA‐binding motif is located near the carboxyl‐terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.

Cooling Prevents Induction of Corneal Damage by Argon Laser Peripheral Iridotomy

PurposeThere are many reports of corneal complications caused by argon laser peripheral iridotomy. In this study, we investigated whether cooling the anterior ocular segment during laser iridotomy prevented corneal damage.MethodsA space for cooling the anterior ocular segment by perfusion with ice-cold water was made between the cornea and a contact lens. Dutch pigmented rabbits were excessively irradiated by an argon green laser, aiming at the peripheral iris. We used a contact lens without a cooling system as a control. Temperature in the anterior chamber and intraocular pressure were also monitored throughout the experiment.ResultsDuring laser treatment, the temperature without the cooling system rose to a maximum of 44.5°C in the anterior chamber, whereas use of the cooling system consistently kept this temperature at 11.1°–16.1°C. Although most eyes treated without cooling showed corneal damage, damage was seen in only a few or in no eyes cooled during laser treatment.ConclusionsArgon laser treatment using contact lenses with a cooling system drastically reduced the corneal damage induced by argon laser peripheral iridotomy. This technique may be acceptable for clinical use, considering its technical simplicity and low incidence of treatment-related complications. Jpn J Ophthalmol 2007;51:317–324

Macroaneurysm on the optic disc in a patient with aortic dissection.

Purpose: We report a case of macroaneurysm on the optic disc, a rare location, accompanied by vitreous hemorrhage in a patient with aortic dissection. Methods: A 60-year-old female with a history of aortic dissection at the age of 51 presented with visual disturbance owing to vitreous hemorrhage in her right eye. During vitrectomy, we found a large macroaneurysm on the optic disc that was beating and oozing blood. However, the macroaneurysm was not treated. Results: The macroaneurysm gradually shrunk, and the beating and oozing of blood disappeared accordingly. Conclusion: Up to now, there have been no reports of macroaneurysm on the optic disc in aortic dissection patients. The association between macroaneurysm on the optic disc and aortic dissection is unclear; therefore, additional case reports may be necessary. To the best of our knowledge, our case is the first one reported in the literature.

Effect of oral tranexamic acid on macular edema associated with retinal vein occlusion or diabetes

Purpose Tranexamic acid (TXA) is a widely used antifibrinolytic agent that can also cause a decrease in vascular permeability. We hypothesized that TXA could improve macular edema (ME) that is caused by an increase in retinal vascular permeability. The aim of this study is to evaluate the efficacy of oral TXA for ME associated with retinal vein occlusion (RVO) or diabetic ME (DME). Patients and methods Oral TXA (1,500 mg daily for 2 weeks) was administered to patients with persistent ME secondary to RVO (7 eyes) and DME (7 eyes). After 2 weeks (ie, the final day of administration) and 6 weeks (ie, 4 weeks after the final administration), best-corrected visual acuity and central macular thickness (CMT) were measured and compared with baseline. Analyses were performed for RVO and DME cases. No other treatment was performed during the study period. Results In RVO cases, significant improvement in CMT was found between baseline (467.7±121.4 μm) and 2-week measurements after treatment (428.7±110.5 μm, p=0.024). No significant change was found in CMT between measurements taken at baseline and 6 weeks after treatment. In DME cases, no significant change was found in CMT between measurements taken at baseline and 2 or 6 weeks after treatment. In all analyses of best-corrected visual acuity, no significant change was observed. Conclusion The results support the hypothesis that plasmin plays a role in the development of ME associated with RVO, and oral TXA administration may be useful as an adjuvant treatment when combined with other agents such as anti-vascular endothelial growth factor.

Valsalva-Like Retinopathy Spontaneously Occurred after Ocular Massage

Purpose: The aim of this study is to describe a case of Valsalva-like retinopathy that occurred after ocular massage. Case Presentation: A healthy 44-year-old Japanese female had massaged her eye with strong pressure several times. Subsequently, she noticed a loss in the left central vision. A left-eye fundus examination showed a dense preretinal hemorrhage located under the internal limiting membrane at the posterior pole and a mild vitreous hemorrhage. We performed a neodymium-doped yttrium-aluminium-garnet laser membranotomy to perforate the internal limiting membrane. Her best-corrected visual acuity improved from 0.01 to 1.0. No retinal vascular abnormalities in the macular area were found. Conclusion: Ocular massage can cause Valsalva-like retinopathy.