Yasuo Yanagi

Effect of anti-VEGF antibody on retinal ganglion cells in rats

Aim: Intravitreal injection of anti-vascular endothelial growth factor (VEGF) antibody (bevacizumab, Avastin) has become one of the chief choices for the treatment of macular oedema and neovascular age-related macular degeneration. However, the effect of blocking the VEGF function has not been thoroughly explored in vivo. A previous study has reported that intravitreal injection of bevacizumab had no retinal toxicity on rats; however, bevacizumab is human-specific and does not react with rat VEGF. In this study, the authors examined the effect of anti-rat VEGF antibody and bevacizumab on rat retina in vivo and in vitro, especially focusing on retinal ganglion cells (RGCs). Methods: In vitro, rat RGCs were purified by a two-step immunopanning procedure, and incubated in the presence of VEGF, bevacizumab, anti-rat VEGF antibody, and control-IgG for three days. The number of viable RGCs was counted. In vivo, after intravitreal injections of bevacizumab, anti-rat VEGF antibody, and control-IgG, viable RGCs were visualised by retrolabelling with Fluo-gold and enumerated to examine the toxicity. Results: In vivo, the mean (standard deviation) number of viable RGCs in the VEGF-treated group (0.99 (0.29) vs control), the bevacizumab-treated group (1.0 (0.23) vs control), the anti-rat VEGF antibody-treated group (0.98 (0.18) vs control) and the control IgG-treated group (0.98 (0.19) vs control) was not statistically different from that of the control group after 3 days. In vitro, the mean (SD) number of viable RGCs in the bevacizumab-treated group (2613 (230)/mm2), the anti-rat VEGF antibody-treated group (2600 (140)/mm2) and the control IgG-treated group (2656 (150)/mm2) was not statistically different from that of the control group (2656 (150)/mm2) after 7 days. There were no apparent histological abnormalities. Conclusion: This study suggests that bevacizumab and anti-rat VEGF antibody have no short-term, direct retinal toxicity using the rat model. Intravitreal injection of bevacizumab shows no short-term, direct toxicity on RGCs.

Background Comparison of Typical Age-related Macular Degeneration and Polypoidal Choroidal Vasculopathy in Japanese Patients

OBJECTIVE To compare background factors of the 2 most dominant subtypes of exudative age-related macular degeneration (AMD) in the Japanese population: typical AMD and polypoidal choroidal vasculopathy (PCV). DESIGN Cross-sectional comparison. PARTICIPANTS Consecutive patients with typical AMD (n = 89) and PCV (n = 138) for the primary survey. For the secondary survey, the number of participants was extended to include 148 typical AMD and 170 PCV patients. All the patients included in the present study had been followed up at The University of Tokyo Hospital outpatient macular clinic. METHODS Background data on gender; age; body mass index; smoking; alcohol consumption; and histories of hypertension, diabetes mellitus (DM), hyperlipidemia, ischemic heart disease, stroke, intensive light exposure, central serous chorioretinopathy (CSC), cataract surgery, glaucoma, and steroid use were obtained mainly through interview. The interviewers were masked to the subtype diagnosis of AMD. Univariate and multivariate logistic regression analyses were performed to identify differences in the background factors between typical AMD and PCV. In the secondary survey, the association of a history of CSC and PCV was confirmed further, and funduscopic findings of an atrophic retinal pigment epithelial (RPE) tract and focal photocoagulation scars that could indicate a history of CSC were investigated. MAIN OUTCOME MEASURES Frequency and mean of background factors in patients with typical AMD or PCV. RESULTS The 2 groups showed similar backgrounds with the exception of their histories of DM and CSC. A history of DM was more frequent in typical AMD (24.7% vs. 13.0% in the primary survey; P = 0.027), whereas a history of CSC was more prevalent in PCV (3.4% vs. 14.7% in the secondary survey; P = 0.0005). Funduscopic findings of an atrophic RPE tract or focal photocoagulation scars were found more frequently in PCV (0.7% vs. 7.6%; P = 0.002). CONCLUSIONS Background factors of typical AMD and PCV are similar but not identical. A history of DM and CSC are more frequent in typical AMD and PCV, respectively.

Retraction: ‘A cell cycle‐dependent co‐repressor mediates photoreceptor cell‐specific nuclear receptor function’

Photoreceptor cell‐specific nuclear receptor (PNR) (NR2E3) acts as a sequence‐specific repressor that controls neuronal differentiation in the developing retina. We identified a novel PNR co‐repressor, Ret‐CoR, that is expressed in the developing retina and brain. Biochemical purification of Ret‐CoR identified a multiprotein complex that included E2F/Myb‐associated proteins, histone deacetylases (HDACs) and NCoR/HDAC complex‐related components. Ret‐CoR appeared to function as a platform protein for the complex, and interacted with PNR via two CoRNR motifs. Purified Ret‐CoR complex exhibited HDAC activity, co‐repressed PNR transrepression function in vitro, and co‐repressed PNR function in PNR target gene promoters, presumably in the retinal progenitor cells. Notably, the appearance of Ret‐CoR protein was cell‐cycle‐stage‐dependent (from G1 to S). Therefore, Ret‐CoR appears to act as a component of an HDAC co‐repressor complex that supports PNR repression function in the developing retina, and may represent a co‐regulator class that supports transcriptional regulator function via cell‐cycle‐dependent expression.

Effects of yellow intraocular lenses on light-induced upregulation of vascular endothelial growth factor

PURPOSE: To investigate the protective effect of a blue‐light filtering intraocular lens (yellow IOL) (YA60BB, Hoya) and an ultraviolet (UV)‐absorbing IOL (VA60BB, Hoya) on light‐induced phototoxicity to retinal pigment epithelial (RPE) cells laden with the lipofuscin fluorophore A2E and on the production of vascular endothelial growth factor (VEGF) after light exposure. SETTING: University of Tokyo, Tokyo, Japan. METHODS: The A2E‐laden ARPE‐19 cells were exposed to white light and a UV‐absorbing IOL or a blue‐light filtering IOL was placed over the light beam. After 48 hours of irradiation, the viability of the cells was determined with WST‐1 (a sodium salt of 4‐[3‐(4‐iodophenyl)‐2‐(4‐nitrophenyl)‐2H‐5‐tetrazolio]‐1,3‐benzene disulfonate) assay, and the secreted protein level of VEGF was determined by enzyme‐linked immunosorbent assay. RESULTS: Without an IOL, the white‐light exposure decreased cell viability to 28% of the nonirradiated control. Although the UV‐absorbing IOL tended to reduce light‐induced cell death, the decrease was not significant. However, the presence of the blue‐light filtering IOL significantly attenuated light‐induced cell damage, increasing cell viability to 42%. The secreted VEGF protein level increased 3.2‐fold after the A2E‐laden RPE cells were exposed to white light. In the presence of the UV‐absorbing IOL, the VEGF protein level decreased, but not significantly. The presence of the blue‐light filtering IOL significantly attenuated the upregulated VEGF expression compared to upregulation without an IOL. CONCLUSION: This study supports the theory that a blue‐light filtering IOL may be more protective against A2E‐induced photochemical damage and inhibit more light‐induced VEGF production than a conventional UV‐absorbing IOL.

Glutathione Peroxidase 4 is Required for Maturation of Photoreceptor Cells

Background: The essentiality of an antioxidant enzyme for the development and maturation of photoreceptor cells has remained unclear. Results: While GPx4-abrogated photoreceptor cells develop and differentiate into rod and cone cells, their outer segments are structurally disorganized and they undergo rapid apoptosis in vivo. Conclusion: GPx4 is essential for the maturation of photoreceptor cells. Significance: A novel role of an antioxidant enzyme for photoreceptor cells is disclosed in this study. Oxidative stress is implicated in the pathologies of photoreceptor cells, and the protective role of antioxidant enzymes for photoreceptor cells have been well understood. However, their essentiality has remained unknown. In this study we generated photoreceptor-specific conditional knock-out (CKO) mice of glutathione peroxidase 4 (GPx4) and showed the critical role of GPx4 for photoreceptor cells. In the wild-type retina the dominant GPx4 expression was in the mitochondria, indicating the mitochondrial variant was the major GPx4 in the retina. In the GPx4-CKO mice, although photoreceptor cells developed and differentiated into rod and cone cells by P12, they rapidly underwent drastic degeneration and completely disappeared by P21. The photoreceptor cell death in the GPx4-CKO mice was associated with the nuclear translocation of apoptosis-inducing factor (AIF) and TUNEL-positive cells. Photoreceptor cells before undergoing apoptosis (P11) exhibited decreased mitochondrial biomass, decreased number of connecting cilia, as well as disorganized structure of outer segments. These findings indicate that GPx4 is a critical antioxidant enzyme for the maturation and survival of photoreceptor cells.

Photodynamic therapy for corneal neovascularization using polymeric micelles encapsulating dendrimer porphyrins

PURPOSE To investigate the accumulation of new photosensitizers (PSs), dendrimer porphyrin (DP, free DP), and DP encapsulation into polymeric micelles (DP-micelle) and the efficacy of photodynamic therapy (PDT) in an experimental corneal neovascularization model in mice. METHODS Corneal neovascularization was induced by suturing 10-0 nylon 1 mm away from the limbal vessel in C57BL6/J mice. To determine the accumulation of free DP and DP-micelle, 10 mg/kg free DP or DP-micelle was administered by intravenous injection 4 days after suture placement. Mice were killed 1, 4, 24, and 168 hours after the injection of PS. Twenty-four hours after the administration of free DP or DP-micelle, mice were treated with a diode laser of 438-nm wavelength at 10 or 50 J/cm(2). Fluorescein angiography was performed before and 7 days after irradiation, and the area of corneal neovascularization was quantified. RESULTS Free DP and DP-micelle accumulated in the neovascularized area 1 hour to 24 hours after administration. Fluorescence of DP was weaker than that of DP-micelle. Neither DP-micelle nor DP could be detected in normal limbal vasculature. In the PDT experiments using PS, mean residual rates of corneal neovascularization were 10.1% in the mice treated with DP-micelle and 21.6% in the mice treated with free DP at 10 J/cm(2) (P < 0.01). At 50 J/cm(2), mean residual rates of corneal neovascularization were 10.6% in the mice treated with DP-micelle and 13.7% in the mice treated with free DP (P > 0.05). Although corneal neovascularization in PDT-treated mice exhibited significant regression compared with the control group, significant energy-related vessel regression with increasing laser energy could not be observed. CONCLUSIONS PDT with DP-micelle and free DP can provide efficacious treatment of corneal neovascularization.

A2E, a Pigment of the Lipofuscin of Retinal Pigment Epithelial Cells, Is an Endogenous Ligand for Retinoic Acid Receptor

Lipofuscin contains fluorophores, which represent a biomarker for cellular aging. Although it remains unsubstantiated clinically, experimental results support that the accumulation of lipofuscin is related to an increased risk of choroidal neovascularization due to age-related macular degeneration, a leading cause of legal blindness. Here, we report that a major lipofuscin component, A2E, activates the retinoic acid receptor (RAR). In vitro experiments using luciferase reporter assay, competitional binding assay, analysis of target genes, and chromatin immunoprecipitation (ChIP) assay strongly suggest that A2E is a bona fide ligand for RAR and induces sustained activation of RAR target genes. A2E-induced vascular endothelial growth factor (VEGF) expression in a human retinal pigment epithelial cell line (ARPE-19) and RAR antagonist blocked the up-regulation of VEGF. The conditioned medium of A2E-treated ARPE-19 cells induced tube formation in human umbilical vascular endothelial cells, which was blocked by the RAR antagonist and anti-VEGF antibody. These results suggest that A2E accumulation results in the phenotypic alteration of retinal pigment epithelial cells, predisposing the environment to choroidal neovascularization development. This is mediated through the agonistic function of A2E, at least in part. The results of this study provide a novel potential therapeutic target for this incurable condition.

Surgically-induced inflammation with 20-, 23-, and 25-gauge vitrectomy systems: an experimental study.

Purpose: To evaluate postoperative intraocular inflammation induced in rabbit eyes subjected to surgery using different vitrectomy systems. Methods: The 20-, 23-, and 25-gauge vitretomy were performed on a total of 12 rabbit eyes, divided into 3 respective groups, and 4 rabbit eyes were used as a normal control group. The surgery consisted of posterior vitreous detachment induction and core vitretomy. The main outcome measures were operative time, volume of balanced saline solution consumed and the intravitreal total protein concentration, determined by bicinchoninic acid protein assays, obtained by vitreous tap on postoperative days 1, 3, and 7. Results: The intravitreal protein averages in the 20-, 23-, and 25-gauge vitretomy groups were, respectively, 31 ± 4.1, 21 ± 2.3, and 7 ± 3.4 mg/mL on postoperative Day 1 (0.13 ± 0.02 mg/mL in the control eye). The intravitreal protein concentration level was significantly lower with the 25-gauge than with 20- or 23-gauge vitrectomy (P < 0.001), and there were no significant differences in intravitreal protein between the 20- and 23-gauge system on postoperative days 1 and 3. At 7 days postoperatively, there were no significant differences in the intravitreal protein levels among the three groups. Significantly less balanced saline solution was consumed in the 25-gauge vitrectomy group (P < 0.001). Conclusion: Postoperative intraocular inflammation can vary among vitrectomy systems. A smaller gauge can minimize the inflammation associated with vitrectomy.

Effects of perfluorocarbon liquids and silicone oil on human retinal pigment epithelial cells and retinal ganglion cells.

Purpose: To examine the effects of perfluorocarbon liquid (PFCL) and silicone oil (SO) on human retinal pigment epithelium (RPE) cells and retinal ganglion cells (RGCs) in vitro. Methods: Human RPE cells (ARPE-19 cells), seeded on microporous inserts, were exposed to PFCL or SO and incubated for 3 or 7 days. Perfluorocarbon liquid was in contact with cells at the apical or baso-lateral side, not inhibiting cell feeding. Then, the quantification of cell proliferation and cell viability were evaluated by WST-1 assay. In the same way, RGCs were exposed for 1 hour or 3 days, and the number of viable RGCs was counted by using a fluorescence viability agent. Results: Perfluorocarbon liquid affected the survival of ARPE-19 cells and RGCs when compared with the nontreated control group. ARPE-19 cells decreased significantly after being in contact with PFCL at the baso-lateral side for 7 days. However, PFCL contact at the apical side reduced the number of RGCs in a time-dependent manner. In case of SO, the viability of the ARPE-19 cells decreased significantly after being in contact with SO at the baso-lateral side for 7 days. However, SO did not reduce the number of RGCs after a 3-day exposure. Conclusion: Perfluorocarbon liquid is directly toxic to ARPE-19 cells when exposed to the cells for 7 days. On the contrary, it seems that RGCs are damaged in a time-dependent manner by the more mechanical rather than toxic effects of PFCL. Silicone oil seems to exert mechanical rather than toxic effects on ARPE-19 cells. When PFCL is used as a postoperative tamponade clinically, understanding the difference in the effects will lead to more effective and safer results.

Inhibition of choroidal neovascularization by blocking vascular endothelial growth factor receptor tyrosine kinase.

PurposeTo investigate the role played by receptors of vascular endothelial growth factors, Flt-1 and KDR/Flk-1, on an experimental model of choroidal neovascularization (CNV).MethodsThe vascular endothelial growth factor-A (VEGF-A) receptor-specific tyrosine kinase inhibitor SU5416 was administered to a laser-induced mouse model of CNV. The formation of CNV and the degree of vascular permeability in Flt-1 tyrosine kinase domain-deficient mice were also investigated.ResultsSU5416 reduced vascularity and vascular endothelial cell proliferation, and promoted endothelial cell apoptosis within CNV. Furthermore, the formation of CNV and the degree of vascular permeability were significantly reduced in Flt-1 tyrosine kinase domain-deficient mice, and this effect was enhanced by the administration of SU5416.ConclusionsBoth Flt-1 and KDR/Flk-1 have a significant role in CNV formation. Suppression of apoptosis may be involved in the process.