Takeshi Mizutani

DICER1/Alu RNA dysmetabolism induces Caspase-8–mediated cell death in age-related macular degeneration

Significance Geographic atrophy is a late stage of age-related macular degeneration (AMD) that causes blindness in millions worldwide characterized by death of the retinal pigmented epithelium (RPE). We previously reported that RPE death is due to a deficiency in the enzyme DICER1, which leads to accumulation of toxic Alu RNA. We also demonstrated that Alu RNA causes RPE death by activating an immune platform called the NLRP3 inflammasome. However, the precise mechanisms of RPE death in this disease remained unresolved. The present study indicates that Alu RNA induces RPE death by activating the enzyme Caspase-8 downstream of inflammasome activation and that blocking Caspase-8 rescues RPE degeneration. This implicates apoptosis as the cell death pathway responsible for Alu RNA cytotoxicity, and these findings provide new potential therapeutic targets for this disease. Geographic atrophy, an advanced form of age-related macular degeneration (AMD) characterized by death of the retinal pigmented epithelium (RPE), causes untreatable blindness in millions worldwide. The RPE of human eyes with geographic atrophy accumulates toxic Alu RNA in response to a deficit in the enzyme DICER1, which in turn leads to activation of the NLRP3 inflammasome and elaboration of IL-18. Despite these recent insights, it is still unclear how RPE cells die during the course of the disease. In this study, we implicate the involvement of Caspase-8 as a critical mediator of RPE degeneration. Here we show that DICER1 deficiency, Alu RNA accumulation, and IL-18 up-regulation lead to RPE cell death via activation of Caspase-8 through a Fas ligand-dependent mechanism. Coupled with our observation of increased Caspase-8 expression in the RPE of human eyes with geographic atrophy, our findings provide a rationale for targeting this apoptotic pathway in this disease.

IL-18 is not therapeutic for neovascular age-related macular degeneration

Up to 50 million people worldwide are afflicted with the devastating blinding disease age-related macular degeneration (AMD)1–3. The vast majority of patients have the currently untreatable “dry” or atrophic form of AMD, characterized by NLRP3 inflammasome-driven degeneration of the retinal pigment epithelium (RPE) supportive cell layer4,5. Blockade of the NLRP3 inflammasome is a next-generation therapeutic target in dry AMD; however, it was recently reported that inflammasome-mediated production of IL18 potentially safeguards the retina against the other, often more visually devastating form of AMD, for which dry AMD patients are at greatly increased risk of developing, known as choroidal neovascularization (CNV)6. Therefore, it is essential, prior to initiating inflammasome-targeting clinical trials, to directly and rigorously assess whether modulating IL18 or the NLRP3 inflammasome affects CNV and RPE cell health.

Human IgG1 antibodies suppress angiogenesis in a target-independent manner

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world’s population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice. Here we show bevacizumab suppressed angiogenesis in three mouse models not via Vegfa blockade but rather Fc-mediated signaling through FcγRI (CD64) and c-Cbl, impairing macrophage migration. Other approved humanized or human IgG1 antibodies without mouse targets (adalimumab, alemtuzumab, ofatumumab, omalizumab, palivizumab and tocilizumab), mouse IgG2a, and overexpression of human IgG1-Fc or mouse IgG2a-Fc, also inhibited angiogenesis in wild-type and FcγR humanized mice. This anti-angiogenic effect was abolished by Fcgr1 ablation or knockdown, Fc cleavage, IgG-Fc inhibition, disruption of Fc-FcγR interaction, or elimination of FcRγ-initated signaling. Furthermore, bevacizumab’s Fc region potentiated its anti-angiogenic activity in humanized VEGFA mice. Finally, mice deficient in FcγRI exhibited increased developmental and pathological angiogenesis. These findings reveal an unexpected anti-angiogenic function for FcγRI and a potentially concerning off-target effect of hIgG1 therapies.

Intravenous immune globulin suppresses angiogenesis in mice and humans

Human intravenous immune globulin (IVIg), a purified IgG fraction composed of ~60% IgG1 and obtained from the pooled plasma of thousands of donors, is clinically used for a wide range of diseases. The biological actions of IVIg are incompletely understood and have been attributed both to the polyclonal antibodies therein and also to their IgG (IgG) Fc regions. Recently, we demonstrated that multiple therapeutic human IgG1 antibodies suppress angiogenesis in a target-independent manner via FcγRI, a high-affinity receptor for IgG1. Here we show that IVIg possesses similar anti-angiogenic activity and inhibited blood vessel growth in five different mouse models of prevalent human diseases, namely, neovascular age-related macular degeneration, corneal neovascularization, colorectal cancer, fibrosarcoma and peripheral arterial ischemic disease. Angioinhibition was mediated by the Fc region of IVIg, required FcγRI and had similar potency in transgenic mice expressing human FcγRs. Finally, IVIg therapy administered to humans for the treatment of inflammatory or autoimmune diseases reduced kidney and muscle blood vessel densities. These data place IVIg, an agent approved by the US Food and Drug Administration, as a novel angioinhibitory drug in doses that are currently administered in the clinical setting. In addition, they raise the possibility of an unintended effect of IVIg on blood vessels.

Recovery of retinal pigment epithelium correlating with restoration of retinal sensitivity in eyes with a retinal pigment epithelial tear

presented with visual acuity (VA) of 20 ⁄200 in the right eye. The patient had undergone photodynamic therapy (PDT) combined with sub-Tenon’s triamcinolone acetonide three times to treat neovascular age–related macular degeneration (AMD) in the right eye. Four months after the last PDT application, fluorescein and indocyanine green angiography (FA ⁄ IA) (HRA2, Heidelberg Engineering, Heidelberg, Germany) showed active occult choroidal neovascularization (CNV) and a large, dome-shaped PED on SDOCT (Cirrus HD-OCT, Carl Zeiss Meditec, Dublin, CA) (Fig. 1). One month later, a retinal pigment epithelial (RPE) tear occurred (Fig. 1). The Fundus autofluorescence (FAF) image on HRA2 showed hypoautofluorescence corresponding to the RPE defect with autofluorescent lipofuscin granules, while hyperautofluorescence was seen in the curled RPE layer (Fig. 2). Interestingly, the hypoautofluorescent area reflecting the denuded lesion secondary to the RPE tear gradually decreased, suggesting that the RPE might regenerate and ⁄or migrate from the border of the RPE tear. Microperimetry on MP-1 (Nidek Technologies, Padova, Italy) showed no recordable retinal sensitivity in the area without an RPE layer. Also, the visual acuity remained poor (20 ⁄ 500) at final visit. However, the retinal sensitivity gradually improved with repopulation of the RPE rather than reattachment of the RPE layer (Figs 1 and 2). An 83-year-old man presented with decreased VA of 20 ⁄ 30 in the left eye. Retinal examination showed a domeshaped PED involving the macula (Fig. 3). IA showed hypofluorescence in the area with the PED and hyperfluorescence from CNV at the edge of the PED (Fig. 3). Three months later, the VA decreased to 20 ⁄ 100 because of a RPE tear. OCT showed a PED with focal disruption of the RPE layer (Fig. 3). FA showed hyperfluorescence in the area with the RPE defect (considered a window defect) and hypofluorescence in the area with the retracted RPE layer. FAF showed a large hypoautofluorescent area associated with no RPE layer (Fig. 4). However, 3 months after the RPE tear, the macular detachment resolved on OCT (Fig. 3). FA (Fig. 3) and FAF (Fig. 4) showed that the area without an RPE layer gradually decreased with recovery of the RPE. The FAF images showed RPE layer regrowth despite a subretinal haemorrhage 4 months after the RPE tear (Fig. 4). However, the visual acuity remained poor (20 ⁄ 500) at final visit. Comment. A RPE tear is a serious complication in eyes with serous pigment epithelial detachment (PED) in neovascular AMD. Consistent with previous report (Chan et al. 2010), there were substantial PED heights prior to the development of RPE tears in both of our cases. Using fundus photograph, FA ⁄ IA, SD-OCT and FAF, we studied two cases with repopulation of the RPE layer and also the recovery of retinal sensitivity onto the denuded area after a RPE tear secondary to neovascular AMD. We cannot rule out the recovery of hyperautofluorescence to be partially related to the presence of fluorophores other than lipofuscin, for example, collagen 1–4, elastin and melanin, etc., although the spectrum of a strongly emitting fluorophore such as lipofuscin likely obscures the spectra of other weakly emitting fluorophores. The background hyperfluorescence on FAF is mainly derived from lipofuscin granules in the RPE cells. FAF was useful for evaluating changes in the RPE defect. The area with the RPE defect was detected as hypofluorescent, and the curled RPE layer was hyperautofluorescent. Gass (1984) described a bare Bruch’s membrane covered by fibrovascular tissues secondary to proliferation of RPE cells at the border of the RPE tear and newly proliferating, nonpigmented RPE cells that did not contain lipofuscin granules. If this is the case, regrowth of RPE cells might be hardly detectable on FAF images. In the current cases, however, FAF images distinctly showed a reduced area of hypofluorescence resulting from lipofuscin covering the RPE cells. Diagnosis and Therapy in Ophthalmology

Tissue Plasminogen Activator as an Antiangiogenic Agent in Experimental Laser-Induced Choroidal Neovascularization in Mice

Purpose We investigate the antiangiogenic efficacy of tissue plasminogen activator (tPA) on experimental laser-induced choroidal neovascularization (CNV) in mice. Methods After CNV was induced by laser photocoagulation in 92 C57BL/6J wild-type mice, tPA (4 or 40 international units [IU]/μl) or PBS was injected intravitreally immediately after laser injury. Fluorescein angiography was performed on day 7 to grade CNV leakage. The CNV volume was measured by confocal microscopy in eyes enucleated 7 days after laser injury. Immunohistochemical studies were performed 3 days after laser injury to evaluate fibrin/fibrinogen and CD31 expression. The possible adverse effects of tPA were assessed by electroretinography (ERG) and histology on day 7. Results Intravitreal administration of tPA significantly suppressed CNV leakage and CNV volume in a dose-dependent manner (P < 0.01). Intravitreal injection of tPA suppressed fibrin/fibrinogen and CD31 expression in laser-induced lesions. Histologic examination and ERG showed no evidence of retinal toxicity in eyes injected with tPA. Conclusions Intravitreal injection of tPA suppressed fibrin/fibrinogen expression and laser-induced CNV. The current results suggested that tPA may be a potential therapeutic adjuvant for treating CNV.

Six-month results of intravitreal ranibizumab for macular edema after branch retinal vein occlusion in a single-center prospective study: visual outcomes and microaneurysm formation

Purpose The aim of this study is to report the 6-month results after one intravitreal ranibizumab (IVR) injection followed by pro re nata dosing for macular edema (ME) after branch retinal vein occlusion. Patients and methods The inclusion criteria included a minimal patient age of 18 years, 20 letters or more best-corrected visual acuity (BCVA) (Early Treatment Diabetic Retinopathy Study [ETDRS] score, 77 letters or less), and central retinal thickness (CRT) of 250 microns or more. The primary outcome measure was the mean BCVA change from baseline at month 6; the secondary outcomes were mean changes in CRT, residual ME, and microaneurysm formation. Results Twenty patients were enrolled from March 2014 through October 2016 at Nagoya City University Hospital. The baseline mean ETDRS letters and CRT were 63.1 and 500 microns, respectively; mean time from symptom onset to initial therapy was 1.80 months; and mean ETDRS gain and CRT reduction were 15.2 letters and 230 microns, respectively. The percentages of patients with Snellen equivalent BCVAs of 20/40 (70 ETDRS letters) or better and 20/20 (85 ETDRS letters) were 90% and 15%, respectively. Residual ME and microaneurysms were observed in 85% and 35% of patients. Microaneurysm formation was associated with delayed initial therapy. Conclusion Prompt initiation of IVR injection provided a better visual prognosis at month 6 and suppressed the microaneurysm formation.