Aya Iriyama

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.

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.

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.

Vitreomacular interface in typical exudative age-related macular degeneration and polypoidal choroidal vasculopathy.

PURPOSE To investigate the association in Japanese between posterior vitreous attachment and the pathologies of typical age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV), 2 major forms of exudative AMD. DESIGN Retrospective observational case series. PARTICIPANTS A total of 378 eyes from 302 subjects (132 with typical AMD, 126 with PCV, 120 controls) from the University of Tokyo Hospital. METHODS Posterior vitreous detachment (PVD) and vitreomacular adhesion (VMA) were investigated by B-mode ultrasonography and spectral-domain optical coherence tomography (SD-OCT), respectively. The greatest linear dimension (GLD) of initial photodynamic therapy (PDT) in a subset of the patients (n=92) receiving PDT was also investigated. MAIN OUTCOME MEASURES Number of eyes with complete PVD and with VMA. The GLD of initial PDT. RESULTS In typical AMD eyes, the frequency of complete PVD was significantly lower (63 [56.8%] of 111 eyes) than in the controls (52 [70.3%] of 74 eyes, risk ratio [RR] 0.76, P=0.021) and the frequency of VMA tended to be higher (14/115 [12.2%] in typical AMD eyes and 6/86 [7.0%] in the controls, RR 2.15, P=0.099). The frequency of complete PVD [77 [63.1%] of the 122 eyes] and VMA (9/108 [8.3%]) in PCV eyes was the same as the controls (RR 0.91, P=0.415 and RR 1.29, P=0.615). In patients with unilateral exudative AMD, the frequency of complete PVD was lower in typical AMD eyes than in fellow eyes (odds ratio [OR] 0.111, P=0.026) and VMA was observed in 7 (17.5%) and 3 (7.5%) typical AMD and fellow eyes, respectively (OR 2.33, P=0.34), whereas in PCV eyes, the frequency of complete PVD was higher (OR 8.00, P=0.045) and the frequency of VMA was the same as in the fellow eyes (OR 0.80, P=1.00). The GLD of the eyes without complete PVD or with VMA was significantly larger than that in the eyes with complete PVD in typical AMD eyes (P=0.042) and the same as that in the eyes with complete PVD in PCV eyes (P=0.67). CONCLUSIONS There is an association between posterior vitreous attachment and typical AMD. However, this association is not evident in PCV.

Outer retinal tubulation in inherited retinal degenerative disease.

Purpose: To investigate the prevalence and characteristics of outer retinal tubulation (ORT) seen in inherited retinal degenerative diseases. Methods: A total of 354 eyes of 177 patients were examined with spectral domain optical coherence tomography. One hundred and twelve patients had retinitis pigmentosa, 58 patients had cone dystrophy, and 7 patients had the Bietti crystalline dystrophy. The images obtained by horizontal and vertical scans were analyzed to explore the possible presence of ORT, estimate their prevalence, morphologic character, and their location in the retinal layers. Results: With spectral domain optical coherence tomography, ORT was identified in 0 of 112 patients with retinitis pigmentosa, unilaterally in 3 of 58 patients with cone dystrophy, and bilaterally in 5 of 7 patients with the Bietti crystalline dystrophy. Outer retinal tubulation was detected under the fovea, and in the outer nuclear layer, ORT was detected in the Bietti crystalline dystrophy with a significantly higher frequency than in cone dystrophy (P < 0.001). Conclusion: There was a higher rate of ORT in the Bietti crystalline dystrophy among inherited retinal degenerative diseases.

Development of Typical Age-related Macular Degeneration and Polypoidal Choroidal Vasculopathy in Fellow Eyes of Japanese Patients with Exudative Age-related Macular Degeneration

PURPOSE To investigate the development of typical age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV) in fellow eyes of Japanese patients with exudative AMD. DESIGN Retrospective observational consecutive case series. METHODS Two hundred and sixteen Japanese patients were enrolled in this study from the outpatient clinic of the University of Tokyo Hospital. Ninety-one patients had typical AMD and one hundred and twenty-five patients had PCV. The average follow-up period was 33.6 and 25.1 months for typical AMD and PCV patients. RESULTS The cumulative incidence of involvement in fellow eyes with overall exudative AMD, including both typical AMD and PCV, was 3.4% in one year, 9.3% in three years, and 11.3% in five years. It was 3.6%, 7.3%, and 11.2% in typical AMD, and 3.2%, 11.1%, and 11.1% in PCV in one, three, and five years, respectively. Before the development of exudative AMD, patients with typical AMD had a variety of funduscopic findings including retinal pigment epithelium (RPE) atrophy, drusen, drusenoid pigment epithelial detachments (PED), and normal macula. PCV patients, on the other hand, had funduscopic findings of RPE atrophy. Inner choroidal vascular abnormality of vascular network and polypoidal formation was observed in several eyes before the clinical manifestation of exudative changes. CONCLUSIONS Typical AMD and PCV had similar probabilities of involving the fellow eye in unilaterally affected Japanese patients. RPE atrophy was a prevailing finding in fellow eyes of patients who developed PCV. In PCV, choroidal vascular network and polypoidal formation gradually grow before exudative changes.

A2E, a component of lipofuscin, is pro-angiogenic in vivo

A recent study in vitro demonstrated that a major lipofuscin component, A2E, serves as a retinoic acid receptor ligand. The current study investigated the effects of A2E on retinal pigment epithelial (RPE) cells in vivo and was performed to extend the understanding of the effects of A2E. Firstly, subretinal injection of A2E was performed and 3 weeks after the injection, and it was demonstrated that subretinal injection of A2E induced RPE cell death, and concomitant upregulation of vascular endothelial growth factor (VEGF) in the RPE and choroid. The upregulation of VEGF was attenuated by an RARα antagonist. Next we performed laser photocoagulation in mice that accumulated A2E either after subretinal injection, by Ccl2 gene knockout or by aging demonstrated that mice that accumulated A2E in the RPE, which showed higher rates of choroidal neobascularization (CNV) formation after weak laser injury than the controls and the formation of CNV was inhibited by an RARα antagonist in all models tested. The data suggest that A2E accumulation induces RPE cell death, and concomitant increase of VEGF. Accumulation of A2E alone is not sufficient to induce CNV in vivo, but induces the expression of VEGF in RPE and choroid. The mice that accumulated A2E in RPE cells are vulnerable to CNV development via RAR activation, at least in part. J. Cell. Physiol. 220: 469–475, 2009.