Xue Tan

Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E

In this study, we show augmented autophagy in the retinal pigment epithelial cell line ARPE‐19 when cultured in the presence of the lipofuscin pigment A2E. A2E alone does not induce RPE cell death, but cell death was induced in the presence of A2E with the autophagy inhibitor 3‐methyladenine (3MA), with a concomitant increase in the generation of mitochondrial reactive oxygen species. On the other hand, the ATP production capacity of mitochondria was decreased in the presence of A2E, and pharmacological inhibition of autophagy had no additional effects. The altered mRNA expression level of mitochondrial function markers was confirmed by real‐time polymerase chain reaction, which showed that the antioxidant enzymes SOD1 and SOD2 were not reduced in the presence of A2E alone, but significantly suppressed with the addition of 3MA. Furthermore, transmission electron micrography revealed autophagic vacuole formation in the presence of A2E, and inhibition of autophagy resulted in the accumulation of abnormal mitochondria with loss of cristae. Spheroid culture of human RPE cells demonstrated debris accumulation in the presence of A2E, and this accumulation was accelerated in the presence of 3MA. These results indicate that autophagy in RPE cells is a vital cytoprotective process that prevents the accumulation of damaged cellular molecules.

Choroidal neovascularization is inhibited via an intraocular decrease of inflammatory cells in mice lacking complement component C3

In early age-related macular degeneration (AMD), complement component C3 can be observed in drusen, which is the accumulation of material beneath the retinal pigment epithelium. The complement pathways, via the activation of C3, can upregulate the expression of cytokines and their receptors and the recruitment of inflammatory leukocytes, both of which play an important role in the development of choroidal neovascularization (CNV) in exudative AMD. Laser-induced CNV lesions were found to be significantly smaller in C3−/− mice than in wild-type mice. By using flow cytometry, we demonstrated that the proportions of intraocular granulocytes, CD11b+F4/80+Ly6Chi and CD11b+F4/80+Ly6Clo cells, were lower in C3−/− mice than in wild-type mice as early as day 1 after laser injury, and the proportions of granulocytes and three macrophage/monocyte subsets were significantly lower on day 3. In contrast, C3−/− mice had more granulocytes and CD11b+F4/80+Ly6Chi cells in peripheral blood than wild-type mice after injury. Further, the expression levels of Vegfa164 were upregulated in intraocular Ly6Chi macrophages/monocytes of C3−/− mice, but not as much as in wild-type mice. Collectively, our data demonstrate that despite a more pronounced induction of systemic inflammation, inhibition of complement factor C3 suppresses CNV by decreasing the recruitment of inflammatory cells to the lesion.

Effects of posterior vitreous detachment on aqueous humour levels of VEGF and inflammatory cytokines

Purpose To investigate the association of posterior vitreous detachment (PVD) with aqueous levels of vascular endothelial growth factor (VEGF) and other inflammatory cytokines. Methods These are prospective comparative studies. Subjects comprised 98 eyes for VEGF concentration and 80 eyes for other cytokines, which are normal except for cataract. PVD was examined by B-mode ultrasonography, and the subjects were divided into complete PVD group (PVD group) or the other group (without PVD group). At the beginning of cataract surgery, aqueous humour was collected and the concentrations of VEGF and other inflammatory cytokines were determined using ELISA and a multiplex cytokine assay, respectively. The concentrations of these cytokines were compared between the two groups. Results Complete PVD was observed in 56 (57%) eyes for VEGF concentration analysis, and 51 (64%) eyes for the other cytokines analysis. The concentrations of VEGF, adjusted for the average age, axial length and gender distribution, was 47 pg/mL in the PVD group and 72 pg/mL in the without PVD group. The concentrations of IP-10, MCP-1, CXCL13 and CCL11 were 53, 450, 3.8 and 6.0 pg/mL in the PVD group, and 100, 560, 7.0 and 8.4 pg/mL in the without PVD group, respectively. Multiple regression analysis revealed that the logarithmic concentration of VEGF, IP-10, MCP-1, CXCL13 and CCL11 were significantly lower in the eyes with PVD than in those without PVD independently of age, sex and axial length (p=0.01, p=0.002, p=0.009, 0.006 and 0.03, respectively). Conclusions PVD is related to the change in the multiple intraocular inflammatory cytokines.

Changes in multiple cytokine concentrations in the aqueous humour of neovascular age-related macular degeneration after 2 months of ranibizumab therapy

Purpose To determine changes in multiple cytokine concentrations in the anterior chamber during the induction phase of ranibizumab treatment in patients with neovascular age-related macular degeneration (AMD). Methods This prospective study included 48 treatment-naïve neovascular AMD eyes of 48 patients who received three consecutive monthly injections of ranibizumab at the Japan Community Health Care Organization Tokyo Shinjuku Medical Center between November 2010 and August 2012. We collected ~0.2 mL aqueous humour before the first and third (2 months later) injections. Controls were 80 eyes with cataracts without retinal disease. The cytokines C-X-C motif chemokine ligand 1 (CXCL1), interferon-γ-induced protein 10 (IP-10), C-X-C motif chemokine ligand 12 (CXCL12), C-X-C motif chemokine ligand 13 (CXCL13), monocyte chemoattractant protein 1 (MCP-1), CCL11, C-C motif chemokine ligand 11 (CCL11), interleukin-6 (IL-6), interleukin-10 (IL-10) and matrix metalloproteinase 9 (MMP-9) were analysed using multiplex cytokine assays. Results Mean ages of the patients with AMD and controls were 73 and 75 years, respectively, and 31 (65%) and 37 (46%) subjects were men, respectively. Polypoidal choroidal vasculopathy was found in 27 eyes (56%). Mean concentrations of cytokines in aqueous humour in patients with neovascular AMD before the first and third ranibizumab injections were as follows (in pg/mL): CXCL1, 8.4 and 3.3; IP-10, 110 and 55; CXCL12, 480 and 240; CXCL13, 9.2 and 2.6; MCP-1, 620 and 220; CCL11, 7.1 and 2.8; IL-6, 5.9 and 1.6; IL-10, 0.15 and 0.015 (all p<0.0001), and MMP-9, 0.92 and 1.5 (p=0.0216), respectively. Concentrations of all cytokines decreased significantly after two consecutive ranibizumab injections, except for MMP-9, which increased significantly. Conclusions After two monthly consecutive antivascular endothelial growth factor injections, inflammatory cytokine levels in the aqueous humour of the eyes with AMD were strongly suppressed, while MMP-9 levels increased.

Excessive retinol intake exacerbates choroidal neovascularization through upregulated vascular endothelial growth factor in retinal pigment epithelium in mice.

As a part of the visual cycle, all-trans-retinol (all-trans-ROL), the major form of vitamin A in circulating blood, is transported to the retinal pigment epithelium (RPE). All-trans-ROL is essential for normal retina function. However, recent researches have shown that excessive retinol intake can cause increase of all-trans-retinal. This can lead to the accumulation of lipofuscin, which is important in the pathogenesis of retina degeneration disease, such as dry type age-related macular degeneration (AMD). Since there are few reports regarding the involvement of all-trans-ROL in exudative AMD, we investigated the effects of all-trans-ROL in vitro and in vivo. We evaluated vascular endothelial growth factor (VEGF) expression in ARPE-19 cells and THP-1 cells after all-trans-ROL treatment using ELISA and real-time RT-PCR. In-vitro tube formation assay was performed with HUVEC cells using the conditioned medium (CM) obtained from ARPE-19 cells treated with all-trans-ROL. Transcriptional activity of retinoic acid receptor (RAR) was evaluated using luciferase assay. In mice, VEGF expressions were investigated in the retina and RPE/choroid after three weeks of excessive oral retinol intake. Laser-induced choroidal neovascularization (CNV) models were evaluated after they were fed with various doses of retinol. VEGF mRNA expression and VEGF production were significantly increased in all-trans-ROL treated ARPE-19 cells, which were inhibited by an RAR antagonist LE540. In contrast, there were no significant changes in VEGF production in THP-1 cells. Transcriptional activity of RAR was upregulated by all-trans-ROL treatment in ARPE-19 cells. The CM, obtained from ARPE-19 cells treated with all-trans-ROL, induced more capillary-like tube formation than cells treated with control vehicles. In vivo, the high retinol diet group has increased VEGF expression in the RPE/choroid and larger lesion size was induced. Our results suggest that all-trans-ROL is a pro-angiogenic factor. Excessive retinoid intake may be a potential risk factor for exudative AMD.

Widespread choroidal thickening and abnormal midperipheral fundus autofluorescence characterize exudative age-related macular degeneration with choroidal vascular hyperpermeability

Purpose To investigate the clinical findings that characterize exudative age-related macular degeneration (AMD) with choroidal vascular hyperpermeability (CVH). Design Retrospective comparative study. Participants Forty-eight consecutive patients attending the outpatient clinic of Tokyo University Hospital between May 2013 and July 2013. Methods The presence or absence of CVH was determined with indocyanine green angiography performed at the latest visit. When CVH was observed, the eye was categorized as CVH(+) AMD, otherwise it was categorized as CVH(-) AMD. Using high-penetration optical coherence tomography, we measured choroidal thickness at the fovea and at four midperipheral areas (mean choroidal thickness at points on 6- and 9-papilla diameter circles superior, inferior, temporal, and nasal to the fovea). Ultrawide field retinal imaging was used to investigate abnormalities in midperipheral fundus autofluorescence (FAF). Choroidal thickness and the proportion of FAF abnormalities were compared between the CVH(+) AMD and CVH(−) AMD eyes and between eyes with polypoidal choroidal vasculopathy and typical AMD. Multiple regression analysis was used to control for treatment history and other characteristics. Results CVH was observed in 17 cases. Choroidal thickness was higher in the CVH(+) AMD eyes than in the CVH(−) AMD eyes at the fovea (325 μm versus 229 μm, respectively; P=0.0010, t-test), superior point (277 μm versus 215 μm, respectively; P=0.0021, t-test), inferior point (225 μm versus 161 μm, respectively; P=0.0002, t-test), and nasal point (202 μm versus 165 μm, respectively; P=0.042, t-test). The significance was maintained after controlling for possible confounders. The choroid was thicker at the fovea and at the inferior point in polypoidal choroidal vasculopathy than in typical AMD. The rate of midperipheral FAF abnormality was significantly higher in the CVH(+) AMD eyes than in the CVH(−) AMD eyes (82% versus 48%, respectively; P=0.031). Conclusion AMD with CVH is associated with widespread choroidal thickening and peripheral FAF abnormalities.

Color vision abnormality as an initial presentation of the complete type of congenital stationary night blindness

Patients with the complete form of congenital stationary night blindness (CSNB) often have reduced visual acuity, myopia, impaired night vision, and sometimes nystagmus and strabismus, however, they seldom complain of color vision abnormality. A 17-year-old male who was at technical school showed abnormalities in the color perception test for employment, and was referred to our hospital for a detailed examination. He had no family history of color vision deficiency and no other symptoms. During the initial examination, his best-corrected visual acuity was 1.2 in both eyes. His fundus showed no abnormalities except for somewhat yellowish reflex in the fovea of both eyes. Electroretinogram (ERG) showed a good response in cone ERG and 30 Hz flicker ERG, however, the bright flash, mixed rod and cone ERG showed a negative type with a reduced b-wave (positive deflection). There was no response in the rod ERG, either. From the findings of the typical ERG, the patient was diagnosed with complete congenital stationary night blindness. This case underscores the importance of ERG in order to diagnose the cause of a color vision anomaly.

ANGIOGRAPHIC FINDINGS OF RANIBIZUMAB-RESISTANT POLYPOIDAL CHOROIDAL VASCULOPATHY AFTER SWITCHING TO A TREAT-AND-EXTEND REGIMEN WITH INTRAVITREAL AFLIBERCEPT.

Purpose: The aim of this study was to study the angiopathic findings of ranibizumab-resistant polypoidal choroidal vasculopathy after switching to a treat-and-extend regimen with intravitreal aflibercept. Methods: The authors retrospectively reviewed 17 eyes of 17 Japanese patients with polypoidal choroidal vasculopathy (10 men and 7 women, age: 73.8 ± 7.4 years) who were treated with intravitreal aflibercept (2 mg/0.05 mL) injections from February 2013 to August 2014 at Tokyo University Hospital. All patients had switched to aflibercept because their polypoidal choroidal vasculopathy had been refractory to ranibizumab. Results: The mean logMAR best-corrected visual acuity at baseline and after 12 months of therapy was 0.30 ± 0.29 (Snellen equivalent: 20/40) and 0.17 ± 0.26 (20/30) (paired t-test P < 0.001). Visual acuity remained stable in 5 cases (29%), deteriorated in 3 (18%), and improved in 9 (53%). Branching vascular networks persisted in all 17 eyes but shrank in 15 (88%). The mean lesion diameter was 3329 ± 1261 &mgr;m at baseline and 3180 ± 1247 &mgr;m after 12 months (P = 0.0002). Conclusion: A treat-and-extend regimen with intravitreal aflibercept for ranibizumab-resistant patients resulted in branching vascular network shrinkage over a 1-year period.

Choroidal Neovascularization Is Inhibited in Splenic-Denervated or Splenectomized Mice with a Concomitant Decrease in Intraocular Macrophage

Purpose To determine the involvement of sympathetic activity in choroidal neovascularization (CNV) using laser-induced CNV in a mouse model. Methods We investigated changes in the proportions of intraocular lymphocytes, granulocytes, and three macrophage subtypes (Ly6Chi, Ly6Cint, and Ly6Clo) after laser injury in mice using flow cytometry, and evaluated CNV lesion size in mice lacking inflammatory cells. Further, we evaluated the lesion size in mice administered the β3 receptor antagonist, splenic-denervated and splenectomized mice. We also assessed changes in the proportions of intraocular macrophages and peripheral blood monocytes in splenic-denervated and splenectomized mice. Lastly, lesion size was compared between splenic-denervated mice with or without adoptive transfer of macrophages following laser injury. After Ly5.1 mice spleen-derived Ly6Chi cells were transferred into Ly5.2 mice, the proportions of intraocular Ly5.1+Ly6Chi cells were compared. Results In WT mice, the proportion of CD4+ T cells recruited into the eye increased progressively from day 3 to day 7 after laser injury, whereas, intraocular CD8+ T cells did not change significantly. Proportions of B220+ cells, granulocytes, and two subtypes of intraocular macrophages (Ly6Chi and Ly6Clo) peaked at day 3 following laser injury. In contrast, Ly6Cint/loCD64+ subtype showed a significantly higher percentage at day 7 after laser injury. There were no differences in lesion size between CD4–/–or Rag2–/–mice and controls, whereas lesion size was significantly reduced in CCR2−/− mice and clodronate liposome-treated mice. CNV lesion area was significantly reduced in mice with β3 blocker treatment, splenic-denervated and splenectomized mice compared with controls. Intraocular Ly6Chi macrophages were also reduced by splenic denervation or splenectomy. Adoptive transfer of spleen-derived Ly6Chi cells increased the lesion size in splenic-denervated mice. Compared with controls, intraocular donor-derived Ly6Chi cells recruited into the eye were reduced in splenic-denervated and splenectomized mice. Conclusions Although lymphocytes had little effect on CNV formation, Ly6Chi macrophages/monocytes exacerbated CNV in mice. Sympathetic activity might contribute to CNV via the recruitment of macrophages to the eye.

Visual acuity loss associated with excessive “dry macula” in exudative age-related macular degeneration

Purpose To investigate the correlation between visual acuity and central macular thickness (CMT) and choroidal thickness (CCT) in patients with wet age-related macular degeneration (AMD). Methods In this retrospective analysis, 14 eyes that received >10 ranibizumab injections (based on pro re nata [PRN] regimen) and maintained initial visual acuity gain were analyzed. The following 5 parameters were measured at the foveal center: CMT (distance from the inner limiting membrane [ILM] to Bruch’s membrane); central retinal thickness (CRT; distance from the ILM to the inner limit of the retinal pigment epithelium or subretinal fluid [SRF]); SRF thickness (SRFT); pigment epithelium detachment thickness (PEDT); and CCT. The correlation between the logarithm of the minimum angle of resolution (logMAR) best-corrected visual acuity (BCVA) and the 5 parameters was examined with generalized estimating equations. Results CMT, CRT, and CCT were negatively correlated with logMAR BCVA (P=0.031, 0.023, and 0.036, respectively) when only CMT values less than the thickness that maximized visual acuity for each eye were used for the analysis. Each 100-μm reduction in CMT, CRT, or CCT improved logMAR BCVA by −0.1, −0.08, or −0.07, respectively. SRFT and PEDT were not correlated with BCVA. The median CMT that maximized the visual acuity was 230 μm. Conclusion Dry macula with CMT <230 μm was associated with temporary decrease in visual acuity in AMD patients whose visual acuity was maintained with PRN regimen.