RESUMEN
Choroidal neovascularization (CNV) is the principal driver of blindness in neovascular age-related macular degeneration (nvAMD). Increased activity of telomerase, has been associated with endothelial cell proliferation, survival, migration, and invasion in the context of tumor angiogenesis. Expanding on this knowledge, we investigated the role of telomerase in the development of CNV in mouse model. We observed increased gene expression and activity of telomerase in mouse CNV. Genetic deficiency of the telomerase components, telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc) suppressed laser-induced CNV in mice. Similarly, a small molecule inhibitor of TERT (BIBR 1532), and antisense oligonucleotides (ASOs) targeting Tert and Terc reduced CNV growth. Bone marrow chimera studies suggested that telomerase activity in non-bone marrow-derived cells is crucial for the development of CNV. Comparison of BIBR 1532 with VEGF neutralizing therapeutic strategy in mouse revealed a comparable level of angiosuppressive activity. However, when BIBR and anti-VEGF antibodies were administered as a combination at sub-therapeutic doses, a statistically significant suppression of CNV was observed. These findings underscore the potential benefits of combining sub-therapeutic doses of BIBR and anti-VEGF antibodies for developing newer therapeutic strategies for NV-AMD. Telomerase inhibition with BIBR 1532 suppressed induction of multiple cytokines and growth factors critical for neovascularization. In conclusion, our study identifies telomerase as a promising therapeutic target for treating neovascular disease of the eye and thus provides a proof of principle for further exploration of telomerase inhibition as a novel treatment strategy for nvAMD.
Asunto(s)
Neovascularización Coroidal , Modelos Animales de Enfermedad , Telomerasa , Telomerasa/antagonistas & inhibidores , Telomerasa/genética , Telomerasa/metabolismo , Animales , Neovascularización Coroidal/patología , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/tratamiento farmacológico , Ratones , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Ratones Endogámicos C57BL , Aminobenzoatos/farmacología , ARN/genética , ARN/metabolismo , Oligonucleótidos Antisentido/farmacología , NaftalenosRESUMEN
Detection of microbial products by multiprotein complexes known as inflammasomes is pivotal to host defense against pathogens. Nucleotide-binding domain leucine-rich repeat (NLR) CARD domain containing 4 (NLRC4) forms an inflammasome in response to bacterial products; this requires their detection by NLR family apoptosis inhibitory proteins (NAIPs), with which NLRC4 physically associates. However, the mechanisms underlying sterile NLRC4 inflammasome activation, which is implicated in chronic noninfectious diseases, remain unknown. Here, we report that endogenous short interspersed nuclear element (SINE) RNAs, which promote atrophic macular degeneration (AMD) and systemic lupus erythematosus (SLE), induce NLRC4 inflammasome activation independent of NAIPs. We identify DDX17, a DExD/H box RNA helicase, as the sensor of SINE RNAs that licenses assembly of an inflammasome comprising NLRC4, NLR pyrin domaincontaining protein 3, and apoptosis-associated speck-like proteincontaining CARD and induces caspase-1 activation and cytokine release. Inhibiting DDX17-mediated NLRC4 inflammasome activation decreased interleukin-18 release in peripheral blood mononuclear cells of patients with SLE and prevented retinal degeneration in an animal model of AMD. Our findings uncover a previously unrecognized noncanonical NLRC4 inflammasome activated by endogenous retrotransposons and provide potential therapeutic targets for SINE RNAdriven diseases.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas de Unión al Calcio/inmunología , ARN Helicasas DEAD-box/inmunología , Inflamasomas/inmunología , ARN/inmunología , Retroelementos/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas de Unión al Calcio/deficiencia , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), produced by cyclic GMP-AMP synthase (cGAS), stimulates the production of type I interferons (IFN). Here we show that cGAMP activates DNA damage response (DDR) signaling independently of its canonical IFN pathways. Loss of cGAS dampens DDR signaling induced by genotoxic insults. Mechanistically, cGAS activates DDR in a STING-TBK1-dependent manner, wherein TBK1 stimulates the autophosphorylation of the DDR kinase ATM, with the consequent activation of the CHK2-p53-p21 signal transduction pathway and the induction of G1 cell cycle arrest. Despite its stimulatory activity on ATM, cGAMP suppresses homology-directed repair (HDR) through the inhibition of polyADP-ribosylation (PARylation), in which cGAMP reduces cellular levels of NAD+; meanwhile, restoring NAD+ levels abrogates cGAMP-mediated suppression of PARylation and HDR. Finally, we show that cGAMP also activates DDR signaling in invertebrate species lacking IFN (Crassostrea virginica and Nematostella vectensis), suggesting that the genome surveillance mechanism of cGAS predates metazoan interferon-based immunity.
Asunto(s)
Daño del ADN , Nucleótidos Cíclicos/metabolismo , Transducción de Señal , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Crassostrea/genética , Crassostrea/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Nucleotidiltransferasas/metabolismo , Fosforilación , Poli ADP Ribosilación , Proteínas Serina-Treonina Quinasas/metabolismo , Reparación del ADN por Recombinación , Anémonas de Mar/genética , Anémonas de Mar/metabolismoRESUMEN
Long interspersed nuclear element-1 (L1)mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNAinduced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNAinduced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.
RESUMEN
Ocular diseases can deteriorate vision to the point of blindness and thus can have a major impact on the daily life of an individual. Conventional therapies are unable to provide absolute therapy for all ocular diseases due to the several limitations during drug delivery across the blood-retinal barrier, making it a major clinical challenge. With recent developments, the vast number of publications undergird the need for nanotechnology-based drug delivery systems in treating ocular diseases. The tool of nanotechnology provides several essential advantages, including sustained drug release and specific tissue targeting. Additionally, comprehensive in vitro and in vivo studies have suggested a better uptake of nanoparticles across ocular barriers. Nanoparticles can overcome the blood-retinal barrier and consequently increase ocular penetration and improve the bioavailability of the drug. In this review, we aim to summarize the development of organic and inorganic nanoparticles for ophthalmic applications. We highlight the potential nanoformulations in clinical trials as well as the products that have become a commercial reality.
RESUMEN
Innate immune signaling through the NLRP3 inflammasome is activated by multiple diabetes-related stressors, but whether targeting the inflammasome is beneficial for diabetes is still unclear. Nucleoside reverse-transcriptase inhibitors (NRTI), drugs approved to treat HIV-1 and hepatitis B infections, also block inflammasome activation. Here, we show, by analyzing five health insurance databases, that the adjusted risk of incident diabetes is 33% lower in patients with NRTI exposure among 128,861 patients with HIV-1 or hepatitis B (adjusted hazard ratio for NRTI exposure, 0.673; 95% confidence interval, 0.638 to 0.710; P < 0.0001; 95% prediction interval, 0.618 to 0.734). Meanwhile, an NRTI, lamivudine, improves insulin sensitivity and reduces inflammasome activation in diabetic and insulin resistance-induced human cells, as well as in mice fed with high-fat chow; mechanistically, inflammasome-activating short interspersed nuclear element (SINE) transcripts are elevated, whereas SINE-catabolizing DICER1 is reduced, in diabetic cells and mice. These data suggest the possibility of repurposing an approved class of drugs for prevention of diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/tratamiento farmacológico , Reposicionamiento de Medicamentos , Inflamasomas/efectos de los fármacos , Resistencia a la Insulina , Inhibidores de la Transcriptasa Inversa/farmacología , Adipocitos/metabolismo , Animales , Supervivencia Celular , ARN Helicasas DEAD-box/metabolismo , Diabetes Mellitus Tipo 2/prevención & control , Dieta Alta en Grasa/efectos adversos , VIH-1/efectos de los fármacos , Hepatitis B , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células Musculares/metabolismo , Ribonucleasa III/metabolismoRESUMEN
Purpose: To determine the effect of voluntary exercise on choroidal neovascularization (CNV) in mice. Methods: Age-matched wild-type C57BL/6J mice were housed in cages equipped with or without running wheels. After four weeks of voluntary running or sedentariness, mice were subjected to laser injury to induce CNV. After surgical recovery, mice were placed back in cages with or without exercise wheels for seven days. CNV lesion volumes were measured by confocal microscopy. The effect of wheel running only in the seven days after injury was also evaluated. Macrophage abundance and cytokine expression were quantified. Results: In the first study, exercise-trained mice exhibited a 45% reduction in CNV volume compared to sedentary mice. In the replication study, a 32% reduction in CNV volume in exercise-trained mice was observed (P = 0.029). Combining these two studies, voluntary exercise was found to reduce CNV by 41% (P = 0.0005). Exercise-trained male and female mice had similar CNV volumes (P = 0.99). The daily running distance did not correlate with CNV lesion size. Exercise only after the laser injury without a preconditioning period did not reduce CNV size (P = 0.41). CNV lesions of exercise-trained mice also exhibited significantly lower F4/80+ macrophage staining and Vegfa and Ccl2 mRNA expression. Conclusions: These findings provide the first experimental evidence that voluntary exercise improves CNV outcomes. These studies indicate that exercise before laser treatment is required to improve CNV outcomes.
Asunto(s)
Neovascularización Coroidal/prevención & control , Actividad Motora , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/irrigación sanguínea , Ribonucleasa III/metabolismo , Animales , Neovascularización Coroidal/genética , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Neovascularización Coroidal/fisiopatología , ARN Helicasas DEAD-box/genética , Humanos , Degeneración Macular/genética , Degeneración Macular/patología , Degeneración Macular/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Neovascularización Retiniana/genética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/parasitología , Neovascularización Retiniana/fisiopatología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Ribonucleasa III/genéticaRESUMEN
Purpose: The misuse of inauthentic cell lines is widely recognized as a major threat to the integrity of biomedical science. Whereas the majority of efforts to address this have focused on DNA profiling, we sought to anatomically, transcriptionally, and functionally authenticate the RF/6A chorioretinal cell line, which is widely used as an endothelial cell line to model retinal and choroidal angiogenesis. Methods: Multiple vials of RF/6A cells obtained from different commercial distributors were studied to validate their genetic, transcriptomic, anatomic, and functional fidelity to bona fide endothelial cells. Results: Transcriptomic profiles of RF/6A cells obtained either de novo or from a public data repository did not correspond to endothelial gene expression signatures. Expression of established endothelial markers were very low or undetectable in RF/6A compared to primary human endothelial cells. Importantly, RF/6A cells also did not display functional characteristics of endothelial cells such as uptake of acetylated LDL, expression of E-selectin in response to TNF-α exposure, alignment in the direction of shear stress, and AKT and ERK1/2 phosphorylation following VEGFA stimulation. Conclusions: Multiple independent sources of RF/6A do not exhibit key endothelial cell phenotypes. Therefore, these cells appear unsuitable as surrogates for choroidal or retinal endothelial cells. Further, cell line authentication methods should extend beyond genomic profiling to include anatomic, transcriptional, and functional assessments.
Asunto(s)
Coroides/irrigación sanguínea , Células Endoteliales/citología , Vasos Retinianos/fisiología , Animales , Biomarcadores , Western Blotting , Línea Celular , Selectina E/genética , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Marcadores Genéticos/genética , Humanos , Inmunohistoquímica , Macaca mulatta , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Fenotipo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcriptoma/genéticaRESUMEN
The molecular basis of intraocular tuberculosis (TB) is not well understood. In this study, we investigated the role of two constituents of viable Mycobacterium tuberculosis - Early Secreted Antigenic Target-6 (ESAT-6), and mycobacterial RNA- in inflammasome activation in the retinal pigment epithelium (RPE), a key site of inflammation in intraocular TB. We found that ESAT-6 induced caspase-1 activation and inflammasome priming in mouse RPE cells, substantially more in wild-type than in Tlr2/3/4/7/9-/-, Myd88-/- or Nlrp3-/- RPE cells. Sub-retinal ESAT-6 injection resulted in greater RPE degeneration in wild-type than in Nlrp3-/- mice. In human ocular TB tissue sections, NLRP3 staining was noted in retina as well as RPE. Mycobacterial RNA, specifically its double stranded component, also induced caspase-1 activation, and the double stranded RNA was immunolocalized to human ocular TB sections. Our observations suggest that inflammasome activation in RPE by viable M. tuberculosis could potentially contribute to human intraocular TB.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Inflamasomas/inmunología , Mycobacterium tuberculosis/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , ARN Bacteriano/inmunología , ARN Bicatenario/inmunología , Tuberculosis Ocular/inmunología , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Caspasa 1/genética , Caspasa 1/inmunología , Interacciones Huésped-Parásitos , Humanos , Inflamasomas/genética , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , ARN Bacteriano/genética , ARN Bicatenario/genética , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/microbiología , Tuberculosis Ocular/genética , Tuberculosis Ocular/microbiologíaRESUMEN
Geographic atrophy is a blinding form of age-related macular degeneration characterized by retinal pigmented epithelium (RPE) death; the RPE also exhibits 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 mouse models is driven by a noncanonical-inflammasome pathway that activates caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-ß production and gasdermin D-dependent interleukin-18 secretion. Decreased DICER1 levels or Alu-RNA accumulation triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, interferon-ß, and cGAS levels were elevated in the RPE in human eyes with geographic atrophy. Collectively, these data highlight an unexpected role of 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 noninfectious human disease, and identify new potential targets for treatment of a major cause of blindness.
Asunto(s)
Atrofia Geográfica/enzimología , Inflamasomas/metabolismo , Nucleotidiltransferasas/metabolismo , Animales , ARN Helicasas DEAD-box/genética , Humanos , Interferón Tipo I/metabolismo , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Ribonucleasa III/genética , Transducción de SeñalRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Elementos Alu , Proteínas Portadoras/metabolismo , Hierro/toxicidad , Epitelio Pigmentado de la Retina/metabolismo , Animales , Proteínas Portadoras/genética , Caspasa 1/genética , Caspasa 1/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Inflamasomas/metabolismo , Hierro/farmacología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
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.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inflamasomas/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Elementos Alu , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas Portadoras/metabolismo , Caspasa 1/metabolismo , Neovascularización Coroidal/tratamiento farmacológico , Modelos Animales de Enfermedad , Atrofia Geográfica/tratamiento farmacológico , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Hepatitis/tratamiento farmacológico , Hígado/efectos de los fármacos , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Receptores Purinérgicos P2X7/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Inhibidores de la Transcriptasa Inversa/uso terapéuticoRESUMEN
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.
Asunto(s)
Elementos Alu , Apoptosis , Caspasa 8/metabolismo , ARN Helicasas DEAD-box/metabolismo , Proteínas del Ojo/metabolismo , Degeneración Macular/metabolismo , ARN/metabolismo , Ribonucleasa III/metabolismo , Animales , Caspasa 8/genética , ARN Helicasas DEAD-box/genética , Proteínas del Ojo/genética , Humanos , Interleucina-18/genética , Interleucina-18/metabolismo , Degeneración Macular/patología , Ratones , Ratones Noqueados , ARN/genética , Ribonucleasa III/genética , Regulación hacia Arriba/genéticaRESUMEN
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Asunto(s)
Elementos Alu , Atrofia Geográfica/fisiopatología , Inflamasomas/fisiología , Receptores Purinérgicos P2X7/fisiología , Transducción de Señal/fisiología , Receptores Toll-Like/fisiología , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Caspasa 1/metabolismo , Modelos Animales de Enfermedad , Gliburida/farmacología , Humanos , Interleucina-18/metabolismo , Ratones , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Nitrilos/farmacología , Antagonistas del Receptor Purinérgico P2X/farmacología , ARN Mensajero/metabolismo , Epitelio Pigmentado de la Retina/citología , Sulfonas/farmacologíaRESUMEN
Neovascular age-related macular degeneration is a leading cause of irreversible vision loss in the Western world. Cytokine-targeted therapies (such as anti-vascular endothelial growth factor) are effective in treating pathologic ocular angiogenesis, but have not led to a durable effect and often require indefinite treatment. Here, we show that Nutlin-3, a small molecule antagonist of the E3 ubiquitin protein ligase MDM2, inhibited angiogenesis in several model systems. We found that a functional p53 pathway was essential for Nutlin-3-mediated retinal antiangiogenesis and disruption of the p53 transcriptional network abolished the antiangiogenic activity of Nutlin-3. Nutlin-3 did not inhibit established, mature blood vessels in the adult mouse retina, suggesting that only proliferating retinal vessels are sensitive to Nutlin-3. Furthermore, Nutlin-3 inhibited angiogenesis in nonretinal models such as the hind limb ischemia model. Our work demonstrates that Nutlin-3 functions through an antiproliferative pathway with conceivable advantages over existing cytokine-targeted antiangiogenesis therapies.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Imidazoles/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Piperazinas/farmacología , Vasos Retinianos/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Miembro Posterior/irrigación sanguínea , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Isquemia/tratamiento farmacológico , Degeneración Macular/tratamiento farmacológico , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Ratas , Vasos Retinianos/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Kaposi's sarcoma-associated herpesvirus (KSHV) infections of endothelial and B cells are etiologically linked with Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma (PEL), respectively. KS endothelial and PEL B cells carry multiple copies of the nuclear episomal latent KSHV genome and secrete a variety of inflammatory cytokines, including interleukin-1ß (IL-1ß) and IL-18. The maturation of IL-1ß and IL-18 depends upon active caspase-1, which is regulated by a multiprotein inflammasome complex induced by sensing of danger signals. During primary KSHV infection of endothelial cells, acting as a nuclear pattern recognition receptor, gamma interferon-inducible protein 16 (IFI16) colocalized with the KSHV genome in the nuclei and interacted with ASC and procaspase-1 to form a functional inflammasome (Kerur N et al., Cell Host Microbe 9:363-375, 2011). Here, we demonstrate that endothelial telomerase-immortalized human umbilical cells (TIVE) supporting KSHV stable latency (TIVE-LTC cells) and PEL (cavity-based B-cell lymphoma 1 [BCBL-1]) cells show evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1ß and pro-IL-18. Interaction of ASC with IFI16 but not with AIM2 or NOD-like receptor P3 (NLRP3) was detected. The KSHV latency-associated viral FLIP (vFLIP) gene induced the expression of IL-1ß, IL-18, and caspase-1 mRNAs in an NF-κB-dependent manner. IFI16 and cleaved IL-1ß were detected in the exosomes released from BCBL-1 cells. Exosomal release could be a KSHV-mediated strategy to subvert IL-1ß functions. In fluorescent in situ hybridization analyses, IFI16 colocalized with multiple copies of the KSHV genome in BCBL-1 cells. IFI16 colocalization with ASC was also detected in lung PEL sections from patients. Taken together, these findings demonstrated the constant sensing of the latent KSHV genome by IFI16-mediated innate defense and unraveled a potential mechanism of inflammation induction associated with KS and PEL lesions.