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Significance DICER1 processes micro-RNAs into their bioactive forms and metabolizes RNAs from short interspersed nuclear element genetic repeats, principally Alu RNAs in humans. DICER1 deficiency is implicated in retinal pigmented epithelium (RPE) degeneration in atrophic age-related macular degeneration (AMD). Here, we report that three independent mouse models of DICER1 deficiency develop RPE degeneration and aberrant choroidal and retinal neovascularization (CRNV), both hallmarks of advanced AMD. These pathologies were dependent on inflammatory caspases 1 and 11 and the signaling adaptor MyD88. We observed reduced DICER1 abundance in a separate model of spontaneous CRNV and developed an adenoassociated vector-mediated DICER1 delivery construct, which reduced the severity of established spontaneous CRNV. Thus, persistent deficiency in DICER1 results in RPE degeneration and CRNV., Degeneration of the retinal pigmented epithelium (RPE) and aberrant blood vessel growth in the eye are advanced-stage processes in blinding diseases such as age-related macular degeneration (AMD), which affect hundreds of millions of people worldwide. Loss of the RNase DICER1, an essential factor in micro-RNA biogenesis, is implicated in RPE atrophy. However, the functional implications of DICER1 loss in choroidal and retinal neovascularization are unknown. Here, we report that two independent hypomorphic mouse strains, as well as a separate model of postnatal RPE-specific DICER1 ablation, all presented with spontaneous RPE degeneration and choroidal and retinal neovascularization. DICER1 hypomorphic mice lacking critical inflammasome components or the innate immune adaptor MyD88 developed less severe RPE atrophy and pathological neovascularization. DICER1 abundance was also reduced in retinas of the JR5558 mouse model of spontaneous choroidal neovascularization. Finally, adenoassociated vector-mediated gene delivery of a truncated DICER1 variant (OptiDicer) reduced spontaneous choroidal neovascularization in JR5558 mice. Collectively, these findings significantly expand the repertoire of DICER1 in preserving retinal homeostasis by preventing both RPE degeneration and pathological neovascularization.

Geographic atrophy is a blinding form of age-related macular degeneration characterized by death of the retinal pigmented epithelium (RPE). In this disease, the RPE displays evidence of DICER1 deficiency, resultant accumulation of endogenous Alu retroelement RNA, and NLRP3 inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human cell culture and in mouse models is driven by a non-canonical inflammasome pathway that results in activation of caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-β (IFN-β) production and gasdermin D-dependent interleukin-18 (IL-18) secretion. Reduction of DICER1 levelsor accumulation of Alu RNA triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, IFN-β, and cGAS levels are elevated in the RPE of human eyes with geographic atrophy. Collectively, these data highlight an unexpected role for cGAS in responding to mobile element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial damage-induced inflammasome activation, expand the immune sensing repertoire of cGAS and caspase-4 to non-infectious human disease, and identify new potential targets for treatment of a major cause of blindness.

Excess iron induces tissue damage and is implicated in age-related macular degeneration (AMD). Iron toxicity is widely attributed to hydroxyl radical formation through Fenton's reaction. We report that excess iron, but not other Fenton catalytic metals, induces activation of the NLRP3 inflammasome, a pathway also implicated in AMD. Additionally, iron-induced degeneration of the retinal pigmented epithelium (RPE) is suppressed in mice lacking inflammasome components caspase-1/11 or Nlrp3 or by inhibition of caspase-1. Iron overload increases abundance of RNAs transcribed from short interspersed nuclear elements (SINEs): Alu RNAs and the rodent equivalent B1 and B2 RNAs, which are inflammasome agonists. Targeting Alu or B2 RNA prevents iron-induced inflammasome activation and RPE degeneration. Iron-induced SINE RNA accumulation is due to suppression of DICER1 via sequestration of the co-factor poly(C)-binding protein 2 (PCBP2). These findings reveal an unexpected mechanism of iron toxicity, with implications for AMD and neurodegenerative diseases associated with excess iron. journal article research support, n.i.h., extramural research support, non-u.s. gov't 2015 Jun 23 2015 06 11 imported

Nucleoside reverse transcriptase inhibitors (NRTIs) are mainstay therapeutics for HIV that block retrovirus replication. Alu (an endogenous retroelement that also requires reverse transcriptase for its life cycle)–derived RNAs activate P2X7 and the NLRP3 inflammasome to cause cell death of the retinal pigment epithelium in geographic atrophy, a type of age-related macular degeneration. We found that NRTIs inhibit P2X7-mediated NLRP3 inflammasome activation independent of reverse transcriptase inhibition. Multiple approved and clinically relevant NRTIs prevented caspase-1 activation, the effector of the NLRP3 inflammasome, induced by Alu RNA. NRTIs were efficacious in mouse models of geographic atrophy, choroidal neovascularization, graft-versus-host disease, and sterile liver inflammation. Our findings suggest that NRTIs are ripe for drug repurposing in P2X7-driven diseases.

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.

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.

Macular edema is the final common pathway of numerous retinal diseases, and ocular disorders associated with this condition are a major cause of blindness. Fluid and molecular exchanges between the blood and retina are tightly regulated and restricted by the blood-retinal barrier (BRB), which is mainly composed of intercellular junctions both at the levels of capillary endothelial cells (inner BRB) and at the retinal pigment epithelium (outer BRB). When a break occurs in the BRB, whether caused by pathological changes in the intervening cells, by imbalance in hydrostatic/oncotic pressure, or by other mechanical forces, fluid and macromolecules enter the retinal intercellular space, causing retinal edema. When this barrier break occurs at or near the macula, fluid accumulation induces formation of macular edema and vision loss ensues. Common diseases associated with macular edema include diabetic retinopathy, age-related macular edema, retinal venous occlusion, retinopathy of prematurity, intraocular inflammation (uveitis, postsurgical, traumatic), anatomical disturbances of retinal vasculature (macular telangiectasias, macroaneurysms), drug-induced break, and tumors. In this chapter, we review and discuss the cellular and molecular mechanisms of BRB integrity and breakdown.

To assess the therapeutic outcome of selective block of VEGFR1, we have evaluated the activity of a new specific antagonist of VEGFR1, named iVR1 (inhibitor of VEGFR1), in syngenic and xenograft colorectal cancer models, in an artificial model of metastatization, and in laser-induced choroid neovascularization. iVR1 inhibited tumor growth and neoangiogenesis in both models of colorectal cancer, with an extent similar to that of bevacizumab, a monoclonal antibody anti-VEGF-A. It potently inhibited VEGFR1 phosphorylation in vivo, determining a strong inhibition of the recruitment of monocyte-macrophages and of mural cells as confirmed, in vitro, by the ability to inhibit macrophages migration. iVR1 was able to synergize with irinotecan determining a shrinkage of tumors that became undetectable after three weeks of combined treatment. Such treatment induced a significant prolongation of survival similar to that observed with bevacizumab and irinotecan combination. iVR1 also fully prevented lung invasion by HCT-116 cells injected in mouse tail vein. Also, iVR1 impressively inhibited choroid neovascularization after a single intravitreal injection. Collectively, data showed the strong potential of iVR1 peptide as a new anti-tumor and anti-metastatic agent and demonstrate the high flexibility of VEGFR1 antagonists as therapeutic anti-angiogenic agents in different pathological contexts.

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.

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.

Purpose Alu RNAs are endogenous pathogens in age-related macular degeneration (AMD). Currently, efforts to develop diagnostic and prognostic tools are limited. Yet, because AMD is a chronic disease, tools to detect pre-clinical disease and monitor progression are an essential part of an effective and comprehensive therapeutic strategy. We tested plasma Alu RNA as a potential biomarker for AMD. Also, cells can convert Alu RNA into DNA, and as such, we determined Alu DNA content of RPE cells. We also genotyped RPE cells for a panel of known AMD risk single nucleotide polymorphisms, and developed an AMD risk stratification system based on age, genotype, and Alu DNA content. Methods RPE cells were isolated from human donor eyes were harvested within 5-10 hours after death. RPE cells were grown in cell culture for four passages. DNA was extracted and subjected to genotyping for AMD-associated SNPs using TaqMan probes. Plasma sample RNA was extracted with Trizol LS and reverse transcribed. qPCR for DNA performed with standard curves of Alu-specific primers and housekeeping single copy genes; for cDNA, Alu quantified per mL plasma. Results The RPE of AMD donors contain increased quantities of Alu DNA. Our results suggest that Alu DNA content increases as a function of age and disease status, and that genotype also modulates disease risk. Finally, Alu RNA content in plasma was significantly increased in AMD versus age-matched healthy controls. Conclusion These efforts set the stage for the utilization of plasma Alu RNA quantity as a novel mechanism-based biomarker in AMD, and also implicate increased Alu DNA burden as a potential mediator of Alu RNA-induced RPE cell death in AMD.

Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life. DOI: http://dx.doi.org/10.7554/eLife.00324.001, eLife digest The inner surface of the vertebrate eye is lined with a multilayered structure known as the retina. The bottom layer of the retina is composed of rods and cones—neurons that are directly sensitive to light—and is called the photoreceptor layer. Rods function primarily in dim light and provide black-and-white vision, while cones support daytime vision and are responsible for colour perception. Unlike the upper layers of the retina, the photoreceptor layer does not contain blood vessels: oxygen and nutrients are instead provided by a structure just underneath the retina called the choroid. The eye relies on the rods and cones converting light into electrical signals, and the photoreceptor layer must remain free of blood vessels for this process to work properly. If blood vessels extend into the photoreceptor layer from rest of the retina (which is above it) or the choroid (below), they can disrupt the retina and give rise to a condition called age-related macular degeneration, which is a leading cause of irreversible blindness in adults. Within the eye, the development of new blood vessels from pre-existing vessels is stimulated by a protein known as vascular endothelial growth factor A (VEGF-A), while an inhibitor protein called sFLT-1 prevents the growth of new blood vessels in the other tissues of the eye like the cornea. However, it has not been clear what keeps the photoreceptor layer (and also the cells that support the photoreceptor layer) free of blood vessels, and what happens to disrupt this process of vascular demarcation in age-related macular degeneration. Now, Luo et al. reveal that cells in the photoreceptor layer produce sFLT-1, and that the levels of this protein are indeed reduced in people with age-related macular degeneration. Using genetic and pharmacological methods, they show that a reduction in sFLT-1 triggers blood vessels to grow into the photoreceptor layer from above or below. Luo et al. also report two new genetic mouse models in which blood vessels form spontaneously in the photoreceptor layer at an early age, which should prove useful for further research into age-related macular degeneration. DOI: http://dx.doi.org/10.7554/eLife.00324.002

Accumulation of Alu RNA transcripts due to DICER1 deficiency in the retinal pigmented epithelium (RPE) promotes geographic atrophy. Recently we showed that Alu RNA activated the NLRP3 inflammasome, leading to RPE cell death via interleukin-18 (IL-18)-mediated MyD88 signaling. However, the molecular basis for NLRP3 inflammasome activation by Alu RNA is not well understood. We sought to decipher the key signaling events triggered by Alu RNA that lead to priming and activation of the NLRP3 inflammasome and, ultimately, to RPE degeneration by investigating the roles of the purinoreceptor P2X7, the transcription factor NF-κB, and the Toll-like receptors (TLRs) in these processes.Human and mouse RPE cells were transfected with a plasmid encoding an Alu element (pAlu) or an in vitro-transcribed Alu RNA. Inflammasome priming was assessed by measuring NLRP3 and IL18 mRNA levels by real-time quantitative PCR. Using immunoblotting, we assessed NF-κB activation by monitoring phosphorylation of its p65 subunit, and inflammasome activation by monitoring caspase-1 cleavage into its active form. RPE degeneration was induced in mice by subretinal transfection of pAlu or Alu RNA. The NF-κB inhibitor BAY 11-7082, the P2X7 receptor antagonist A-740003, and the NLRP3 inflammasome inhibitor glyburide were delivered by intravitreous injections. We studied wild-type (WT) C57Bl/6J, P2rx7(-/-), Nfkb1(-/-), and Tlr23479(-/-) mice. RPE degeneration was assessed by fundus photography and zonula occludens-1 (ZO-1) staining of mouse RPE.Alu RNA-induced NF-κB activation, independent of TLR-1, -2, -3, -4, -6, -7, and -9 signaling, was required for priming the NLRP3 inflammasome. Nfkb1(-/-) and P2rx7(-/-) mice and WT mice treated with the pharmacological inhibitors of NF-κB, P2X7, or NLRP3, were protected against Alu RNA-induced RPE degeneration.NF-κB and P2X7 are critical signaling intermediates in Alu RNA-induced inflammasome priming and RPE degeneration. These molecules are novel targets for rational drug development for geographic atrophy.

This work was supported by FCT PTDC/SAU-ORG/113617/2009 (www.fct.pt). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors would like to thank other members of the Angiogenesis Lab for their input and suggestions. The Program for Advanced Medical Education is sponsored by Fundacao Calouste Gulbenkian, Fundacao Champalimaud, Ministerio da Saude and Fundacao para a Ciencia e Tecnologia, Portugal. Delta-like 4 (Dll4) is a ligand of the Notch pathway family which has been widely studied in the context of tumor angiogenesis, its blockade shown to result in non-productive angiogenesis and halted tumor growth. As Dll4 inhibitors enter the clinic, there is an emerging need to understand their side effects, namely the systemic consequences of Dll4:Notch blockade in tissues other than tumors. The present study focused on the effects of systemic anti-Dll4 targeting in the bone marrow (BM) microenvironment. Here we show that Dll4 blockade with monoclonal antibodies perturbs the BM vascular niche of sub-lethally irradiated mice, resulting in increased CD31+, VE-Cadherin+ and c-kit+ vessel density, and also increased megakaryocytes, whereas CD105+, VEGFR3+, SMA+ and lectin+ vessel density remained unaltered. We investigated also the expression of angiocrine genes upon Dll4 treatment in vivo, and demonstrate that IGFbp2, IGFbp3, Angpt2, Dll4, DHH and VEGF-A are upregulated, while FGF1 and CSF2 are reduced. In vitro treatment of endothelial cells with anti-Dll4 reduced Akt phosphorylation while maintaining similar levels of Erk 1/2 phosphorylation. Besides its effects in the BM vascular niche, anti-Dll4 treatment perturbed hematopoiesis, as evidenced by increased myeloid (CD11b+), decreased B (B220+) and T (CD3+) lymphoid BM content of treated mice, with a corresponding increase in myeloid circulating cells. Moreover, anti-Dll4 treatment also increased the number of CFU-M and -G colonies in methylcellulose assays, independently of Notch1. Finally, anti-Dll4 treatment of donor BM improved the hematopoietic recovery of lethally irradiated recipients in a transplant setting. Together, our data reveals the hematopoietic (BM) effects of systemic anti-Dll4 treatment result from qualitative vascular changes and also direct hematopoietic cell modulation, which may be favorable in a transplant setting. publishersversion published

Abstract 1321 The Delta:Notch signaling pathway regulates multiple aspects of hematopoietic cell differentiation and function and more recently Delta-like 4 (Dll4) has been shown also to regulate adult neo-vascularization (angiogenesis), namely in tumors, which led to the development of therapeutic strategies targeting Dll4 for the treatment of different cancers. Nevertheless, the current therapeutic strategies to target Dll4, which were suggested to result in non-functional vessels in the tumors, appear also to affect the vasculature of normal organs. Given the importance of the Bone marrow (BM) vascular niche in regulating hematopoiesis, we hypothesized Dll4 targeting might also affect BM function. In the present study, we describe 2 means of targeting Dll4: one using Dll4 specific neutralizing monoclonal antibodies (systemic treatment), and another using VECadCreERT2Dll4lox/lox mice, where Dll4 is specifically ablated on VE-Cadherin expressing cells. Using these 2 models, we investigated if Dll4 targeting affected the BM vascular niche and consequently affected hematopoietic recovery following irradiation and BM recovery in a transplant setting. First, we show that the genetic ablation of Dll4 on VE-Cadherin+ vessels (VECadCreERT2Dll4lox/lox mice) and also anti-Dll4 antibody treatment of WT sub-lethally irradiated mice (300rad) does affect the BM vascular niche, resulting in increased megakaryocytes (CD41+), CD31+ and VE-Cadherin+ vessels, without significant changes on CD105+ vessels. Interestingly, the systemic anti-Dll4 treatment of non-irradiated and sub-lethally irradiated WT mice increased the BM and peripheral blood myeloid (CD11b+) content and promoted hematopoietic progenitor cell (Sca1+Flk1−) mobilization, as determined by FACS analysis. The genetic model (VECadCreERT2Dll4lox/lox mice) showed no changes in hematopoietic cells percentage or mobilization. However, BM recovery in a transplant setting was improved in both models. Lethally irradiated WT mice transplanted with anti-Dll4 treated BM following sub-lethal irradiation or BM from VECadCreERT2Dll4lox/lox mice showed faster hematopoietic recovery than control mice transplanted with WT BM alone. Taken together, we show that Dll4 blockade perturbs the BM vascular niche, increasing VE-Cadherin+ and CD31+ vessel number and also megakaryocyte BM content, both in the setting of Dll4 genetic ablation (using VECadCreERT2Dll4lox/lox mice) and also using a systemic approach to target Dll4. However, these vascular changes do not, at least following sub-lethal irradiation, significantly affect the BM recovery or affect the normal distribution of the hematopoietic populations. Nevertheless, the significant effects observed in a BM transplant setting (both where donor had been exposed to the systemic anti-Dll4 blockade and where Dll4 had been specifically ablated on VE-Cadherin+ vessels) suggest the existence of cell autonomous (and BM vascular niche-autonomous) effects of Dll4 targeting which deserve further detailed analysis. These data also suggest targeting Dll4-expressing vessels in the setting of BM malignancies may have therapeutic benefit, since it appears to have limited effects on normal BM/hematopoietic function. We are currently investigating the therapeutic efficacy of Dll4 targeting using the genetic model and the antibody-based approaches, in the setting of malignant myeloma and also acute leukemias. Disclosures: No relevant conflicts of interest to declare.

An 18-year-old woman presented with a 2-month history of visual loss in the right eye. Visual acuity was 20/100 in the affected eye and 20/20 in the left eye. Ophthalmoscopy revealed an orange–yellow tumor (Panel A, arrow) with tortuous feeding vessels and optic-disk edema (Panel A, arrowhead), indicating retinal hemangioblastoma. The patient underwent ruthenium plaque radiotherapy, cryotherapy, and photodynamic therapy, which induced complete tumor regression and sclerosis of the dilated vessels (Panel B). After treatment, the visual acuity improved, to 20/70. A systemic evaluation, including magnetic resonance imaging of the brain, computed tomography of the abdomen, and measurement of urinary . . .