RESUMEN
Aging of the microcirculatory network plays a central role in the pathogenesis of a wide range of age-related diseases, from heart failure to Alzheimer's disease. In the eye, changes in the choroid and choroidal microcirculation (choriocapillaris) also occur with age, and these changes can play a critical role in the pathogenesis of age-related macular degeneration (AMD). In order to develop novel treatments for amelioration of choriocapillaris aging and prevention of AMD, it is essential to understand the cellular and functional changes that occur in the choroid and choriocapillaris during aging. In this review, recent advances in in vivo analysis of choroidal structure and function in AMD patients and patients at risk for AMD are discussed. The pathophysiological roles of fundamental cellular and molecular mechanisms of aging including oxidative stress, mitochondrial dysfunction, and impaired resistance to molecular stressors in the choriocapillaris are also considered in terms of their contribution to the pathogenesis of AMD. The pathogenic roles of cardiovascular risk factors that exacerbate microvascular aging processes, such as smoking, hypertension, and obesity as they relate to AMD and choroid and choriocapillaris changes in patients with these cardiovascular risk factors, are also discussed. Finally, future directions and opportunities to develop novel interventions to prevent/delay AMD by targeting fundamental cellular and molecular aging processes are presented.
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Envejecimiento/fisiología , Coroides/irrigación sanguínea , Microcirculación/fisiología , Flujo Sanguíneo Regional/fisiología , Vasos Retinianos/patología , Degeneración Macular Húmeda/fisiopatología , Progresión de la Enfermedad , Humanos , Vasos Retinianos/fisiopatología , Degeneración Macular Húmeda/diagnósticoRESUMEN
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
OBJECTIVES: Age-related macular degeneration (AMD) is the leading cause of blindness in the United States. Although AMD shares multiple risk factors with coronary artery disease (CAD), the association between AMD and CAD has not been established. The objective of our study was to demonstrate an association between the diagnosis of AMD and CAD and/or major cardiovascular risk factors. METHODS: We performed a retrospective chart review of >13,000 patients at the Lexington Veterans Affairs Medical Center. Patients diagnosed as having AMD served as cases, and patients diagnosed with cataract and no AMD served as controls. We examined the prevalence of CAD and associated risk factors in both groups using univariate analysis followed by multivariate analyses to examine the association between AMD and CAD after adjusting for known common risk factors. RESULTS: We identified 3950 patients with AMD and 9166 controls. Patients with AMD were on average 6 years older than controls (P < 0.001) and had a significantly higher prevalence of CAD (39% vs 34%) and hypertension (88% vs 83%) but lower incidence of diabetes mellitus and smoking. Estimated odds ratio relating CAD to AMD was 1.22 (95% confidence interval 1.13-1.32; P < 0.001). The association between CAD and AMD remained significant in multivariate analyses in older individuals (76 years and older). When we conducted a secondary analysis and matched the AMD and non-AMD groups based on age, the association between CAD and AMD remained significant (39.4% in the AMD group vs 36.6% in the non-AMD group; P = 0.011). CONCLUSIONS: These findings support the existence of an association between CAD and AMD, particularly in older adult patients in the predominantly male Veterans Affairs population. Such an association between AMD and systemic vascular disease justifies the potential coscreening for these conditions.
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Enfermedad Coronaria/complicaciones , Enfermedad Coronaria/epidemiología , Degeneración Macular/complicaciones , Degeneración Macular/epidemiología , Veteranos/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Humanos , Incidencia , Masculino , Registros Médicos , Medicina Militar , Prevalencia , Estudios Retrospectivos , Factores de Riesgo , Fumar/efectos adversos , Estados Unidos/epidemiologíaRESUMEN
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.
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Neovascularización Coroidal/metabolismo , Degeneración Macular/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Neovascularización Retiniana/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Visión Ocular , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos/farmacología , Estudios de Casos y Controles , Neovascularización Coroidal/genética , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Humanos , Degeneración Macular/genética , Degeneración Macular/patología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células Fotorreceptoras de Vertebrados/patología , Interferencia de ARN , Neovascularización Retiniana/genética , Neovascularización Retiniana/patología , Epitelio Pigmentado de la Retina/patología , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 1 de Factores de Crecimiento Endotelial Vascular/deficiencia , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Monthly intraocular injections are widely used to deliver protein-based drugs that cannot cross the blood-retina barrier for the treatment of leading blinding diseases such as age-related macular degeneration (AMD). This invasive treatment carries significant risks, including bleeding, pain, infection, and retinal detachment. Further, current therapies are associated with a rate of retinal fibrosis and geographic atrophy significantly higher than that which occurs in the described natural history of AMD. A novel therapeutic strategy which improves outcomes in a less invasive manner, reduces risk, and provides long-term inhibition of angiogenesis and fibrosis is a felt medical need. Here we show that a single intravenous injection of targeted, biodegradable nanoparticles delivering a recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the retina and regresses CNV in primate and murine AMD models. Moreover, this treatment suppressed subretinal fibrosis, which is currently not addressed by clinical therapies. Murine vision, as tested by OptoMotry, significantly improved with nearly 40% restoration of visual loss induced by CNV. We found no evidence of ocular or systemic toxicity from nanoparticle treatment. These findings offer a nanoparticle-based platform for targeted, vitreous-sparing, extended-release, nonviral gene therapy.
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ADN/administración & dosificación , Terapia Genética/métodos , Degeneración Macular/terapia , Nanocápsulas/administración & dosificación , Neovascularización Patológica/terapia , Retina/patología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , ADN/genética , Fibrosis , Haplorrinos , Ratones , Resultado del TratamientoRESUMEN
Deficient expression of the RNase III DICER1, which leads to the accumulation of cytotoxic Alu RNA, has been implicated in degeneration of the retinal pigmented epithelium (RPE) in geographic atrophy (GA), a late stage of age-related macular degeneration that causes blindness in millions of people worldwide. Here we show increased extracellular-signal-regulated kinase (ERK) 1/2 phosphorylation in the RPE of human eyes with GA and that RPE degeneration in mouse eyes and in human cell culture induced by DICER1 depletion or Alu RNA exposure is mediated via ERK1/2 signaling. Alu RNA overexpression or DICER1 knockdown increases ERK1/2 phosphorylation in the RPE in mice and in human cell culture. Alu RNA-induced RPE degeneration in mice is rescued by intravitreous administration of PD98059, an inhibitor of the ERK1/2-activating kinase MEK1, but not by inhibitors of other MAP kinases such as p38 or JNK. These findings reveal a previously unrecognized function of ERK1/2 in the pathogenesis of GA and provide a mechanistic basis for evaluation of ERK1/2 inhibition in treatment of this disease.
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Regulación Enzimológica de la Expresión Génica , Degeneración Macular/enzimología , Degeneración Macular/terapia , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Animales , ARN Helicasas DEAD-box/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Humanos , Ratones , Fosforilación , Epitelio Pigmentado de la Retina/metabolismo , Ribonucleasa III/metabolismo , Transducción de SeñalRESUMEN
PURPOSE: To evaluate the safety profile of a brimonidine extended release intravitreal implant, in normotensive rabbit eyes. METHODS: Devices were made from hollow poly-l-lactic acid (PLA) tubes and contained hundred micrograms of brimonidine pamoate. Device was injected intravitreally in one eye of 12 New Zealand pigmented rabbits, whereas other eye was injected with a sham implant in masked fashion. Ocular examination was conducted at baseline and months 1, 3 and 6 including dilated fundus examination and electro-retinogram (ERG). Four rabbits were sacrificed at each time-point for retinal histology. ERG data were compared between groups and time-points using anova. RESULTS: No complications were reported from either eye of any rabbits over a 6-month period. Photopic A wave was reduced in the control eye at 1 month compared with baseline (p < 0.01). There was no significant difference in other ERG parameters between the groups at different time-points. Gross retinal histology was normal at all time-points. CONCLUSION: Extended release intravitreal brimonidine device was found to be safe and in normotensive rabbit eyes.
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Antihipertensivos/toxicidad , Portadores de Fármacos , Quinoxalinas/toxicidad , Cuerpo Vítreo/efectos de los fármacos , Animales , Antihipertensivos/administración & dosificación , Tartrato de Brimonidina , Adaptación a la Oscuridad/fisiología , Evaluación Preclínica de Medicamentos , Implantes de Medicamentos , Electrorretinografía , Masculino , Poliésteres , Quinoxalinas/administración & dosificación , Conejos , Retina/efectos de los fármacos , Retina/fisiologíaRESUMEN
Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.
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Elementos Alu , ARN Helicasas DEAD-box/metabolismo , Atrofia Geográfica/inmunología , Atrofia Geográfica/patología , Inflamasomas/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Ribonucleasa III/metabolismo , Animales , Proteínas Portadoras/metabolismo , Atrofia Geográfica/metabolismo , Humanos , Inflamasomas/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Epitelio Pigmentado de la Retina/patología , Receptores Toll-Like/metabolismoRESUMEN
The discovery of sequence-specific gene silencing by endogenous double-stranded RNAs (dsRNA) has propelled synthetic short-interfering RNAs (siRNAs) to the forefront of targeted pharmaceutical engineering. The first clinical trials utilized 21-nucleotide (nt) siRNAs for the treatment of neovascular age-related macular degeneration (AMD). Surprisingly, these compounds were not formulated for cell permeation, which is required for bona fide RNA interference (RNAi). We showed that these "naked" siRNAs suppress neovascularization in mice not via RNAi but via sequence-independent activation of cell surface Toll-like receptor-3 (TLR3). Here, we demonstrate that noninternalized siRNAs induce retinal degeneration in mice by activating surface TLR3 on retinal pigmented epithelial cells. Cholesterol conjugated siRNAs capable of cell permeation and triggering RNAi also induce the same phenotype. Retinal degeneration was not observed after treatment with siRNAs shorter than 21-nts. Other cytosolic dsRNA sensors are not critical to this response. TLR3 activation triggers caspase-3-mediated apoptotic death of the retinal pigment epithelium (RPE) via nuclear translocation of interferon regulatory factor-3. While this unexpected adverse effect of siRNAs has implications for future clinical trials, these findings also introduce a new preclinical model of geographic atrophy (GA), a late stage of dry AMD that causes blindness in millions worldwide.
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Factor 3 Regulador del Interferón/metabolismo , ARN Interferente Pequeño/toxicidad , Degeneración Retiniana/inducido químicamente , Receptor Toll-Like 3/metabolismo , Animales , Caspasa 3/metabolismo , Muerte Celular/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , ARN Interferente Pequeño/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Epitelio Pigmentado de la Retina/metabolismo , Transducción de SeñalRESUMEN
Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.
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Elementos Alu/genética , ARN Helicasas DEAD-box/deficiencia , Degeneración Macular/genética , Degeneración Macular/patología , ARN/genética , ARN/metabolismo , Ribonucleasa III/deficiencia , Animales , Muerte Celular , Supervivencia Celular , Células Cultivadas , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ratones , MicroARNs/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Fenotipo , Epitelio Pigmentado de la Retina/enzimología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
Retinal vascular disease is the most common cause of macular edema (ME). While there are several etiologies of vascular compromise and subsequent macular leakage, diabetic retinopathy is the most prevalent and continues to challenge ophthalmologists and frustrate patients due to its refractory nature. In response to this epidemic, diabetic ME (DME) along with cystoid ME (CME) have been areas of active investigation both in the clinic and the laboratory. Several decades of basic science research have revealed a growing and complex array of cytokine growth factors and proinflammatory mediators which are capable of inciting the cellular changes that result in accumulation of fluid within the retina. Much of this new molecular foundation provides the current and fundamental scaffold for understanding the pathologic process of ME while simultaneously identifying potential therapeutic targets. Whereas CME has classically been treated with corticosteroids and nonsteroidal antiinflammatory drugs, recent clinical studies have demonstrated improved visual outcomes for DME treatment with light focal/grid laser, corticosteroids and anti-vascular endothelial growth factor antibodies. Yet, each of these treatments has differential effects on the multifactorial mechanisms of ME. This article reviews the anatomical, cellular and molecular derangements associated with ME and highlights specific pathways targeted by current treatments.
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Antiinflamatorios/uso terapéutico , Terapia por Láser/métodos , Enfermedades de la Retina , Vasos Retinianos/patología , Humanos , Pronóstico , Enfermedades de la Retina/diagnóstico , Enfermedades de la Retina/etiología , Enfermedades de la Retina/terapiaRESUMEN
Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993-994).
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Linfangiogénesis/genética , Linfangiogénesis/fisiología , Vasos Linfáticos/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Empalme Alternativo , Animales , Animales Recién Nacidos , Secuencia de Bases , Córnea/irrigación sanguínea , Córnea/crecimiento & desarrollo , Córnea/metabolismo , ADN Complementario/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Factor C de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor C de Crecimiento Endotelial Vascular/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/deficienciaRESUMEN
Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.
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Degeneración Macular/diagnóstico , Degeneración Macular/terapia , Receptores CCR3/antagonistas & inhibidores , Receptores CCR3/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Células Cultivadas , Quimiocina CCL11/antagonistas & inhibidores , Quimiocina CCL11/metabolismo , Quimiocina CCL24/antagonistas & inhibidores , Quimiocina CCL24/metabolismo , Quimiocina CCL26 , Quimiocinas CC/antagonistas & inhibidores , Quimiocinas CC/metabolismo , Coroides/irrigación sanguínea , Coroides/citología , Coroides/metabolismo , Neovascularización Coroidal/diagnóstico , Neovascularización Coroidal/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Inflamación , Leucocitos , Ligandos , Degeneración Macular/metabolismo , Ratones , Ratones Endogámicos C57BL , Puntos Cuánticos , Receptores CCR3/análisis , Receptores CCR3/genética , Receptores CCR3/inmunología , Retina/efectos de los fármacos , Retina/patología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Neovascularization in response to tissue injury consists of the dual invasion of blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessels. We reported recently that 21-nt or longer small interfering RNAs (siRNAs) can suppress hemangiogenesis in mouse models of choroidal neovascularization and dermal wound healing independently of RNA interference by directly activating Toll-like receptor 3 (TLR3), a double-stranded RNA immune receptor, on the cell surface of blood endothelial cells. Here, we show that a 21-nt nontargeted siRNA suppresses both hemangiogenesis and lymphangiogenesis in mouse models of neovascularization induced by corneal sutures or hindlimb ischemia as efficiently as a 21-nt siRNA targeting vascular endothelial growth factor-A. In contrast, a 7-nt nontargeted siRNA, which is too short to activate TLR3, does not block hemangiogenesis or lymphangiogenesis in these models. Exposure to 21-nt siRNA, which we demonstrate is not internalized unless cell-permeating moieties are used, triggers phosphorylation of cell surface TLR3 on lymphatic endothelial cells and induces apoptosis. These findings introduce TLR3 activation as a method of jointly suppressing blood and lymphatic neovascularization and simultaneously raise new concerns about the undesirable effects of siRNAs on both circulatory systems.
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Vasos Linfáticos/metabolismo , Neovascularización Fisiológica , ARN Interferente Pequeño/genética , Receptor Toll-Like 3/metabolismo , Animales , Apoptosis , Proliferación Celular , Células Endoteliales/citología , Miembro Posterior/irrigación sanguínea , Miembro Posterior/metabolismo , Ratones , Fosforilación , Receptor Toll-Like 3/genéticaRESUMEN
Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-alpha/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.
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Terapia Genética/métodos , Inmunidad Innata/inmunología , Neovascularización Patológica/inmunología , Neovascularización Patológica/prevención & control , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/metabolismo , Receptor Toll-Like 3/metabolismo , Animales , Línea Celular , Células Endoteliales/metabolismo , Humanos , Interferón gamma/inmunología , Interleucina-12/inmunología , Degeneración Macular/complicaciones , Degeneración Macular/genética , Degeneración Macular/terapia , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/genética , Neovascularización Patológica/terapia , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Receptor Toll-Like 3/química , Receptor Toll-Like 3/genética , Factor A de Crecimiento Endotelial Vascular/genéticaAsunto(s)
Modelos Animales de Enfermedad , Terapia Genética , Macrófagos/fisiología , Degeneración Macular/terapia , Epitelio Pigmentado Ocular , Envejecimiento , Animales , Quimiocina CCL2/administración & dosificación , Quimiocina CCL2/genética , Vectores Genéticos , Degeneración Macular/genética , Ratones , Ratones Noqueados , Epitelio Pigmentado Ocular/metabolismo , Drusas Retinianas/etiologíaRESUMEN
Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.
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Córnea/irrigación sanguínea , Córnea/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Eliminación de Gen , Ratones , Neovascularización Fisiológica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solubilidad , Trichechus , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/deficiencia , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
VEGF-A promotes angiogenesis in many tissues. Here we report that choroidal neovascularization (CNV) incited by injury was increased by excess VEGF-A before injury but was suppressed by VEGF-A after injury. This unorthodox antiangiogenic effect was mediated via VEGFR-1 activation and VEGFR-2 deactivation, the latter via Src homology domain 2-containing (SH2-containing) tyrosine phosphatase-1 (SHP-1). The VEGFR-1-specific ligand placental growth factor-1 (PlGF-1), but not VEGF-E, which selectively binds VEGFR-2, mimicked these responses. Excess VEGF-A increased CNV before injury because VEGFR-1 activation was silenced by secreted protein, acidic and rich in cysteine (SPARC). The transient decline of SPARC after injury revealed a temporal window in which VEGF-A signaling was routed principally through VEGFR-1. These observations indicate that therapeutic design of VEGF-A inhibition should include consideration of the level and activity of SPARC.
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Inhibidores de la Angiogénesis/metabolismo , Neovascularización Coroidal , Osteonectina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Ojo/anatomía & histología , Ojo/metabolismo , Ojo/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Noqueados , Osteonectina/genética , Factor de Crecimiento Placentario , Proteínas Gestacionales/metabolismo , Proteína Fosfatasa 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal/fisiología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30-50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD.
Asunto(s)
Neovascularización Coroidal/metabolismo , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Degeneración Macular/metabolismo , Drusas Retinianas/metabolismo , Anciano de 80 o más Años , Animales , Neovascularización Coroidal/genética , Complemento C3a/análisis , Complemento C3a/genética , Complemento C5a/análisis , Complemento C5a/genética , Femenino , Humanos , Degeneración Macular/genética , Masculino , Ratones , Ratones Mutantes , Drusas Retinianas/genética , Drusas Retinianas/inmunología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: To detail, by DNA microarrays and cellular structure labeling, the in vitro responses of retinal pigment epithelial (RPE) cells to a nonlethal dose of the oxidant agent hydroquinone (HQ). METHODS: The viability of growth-quiescent ARPE-19 cells after treatment with HQ was measured by XTT conversion, (3)H-leucine incorporation, trypan blue exclusion, and the presence of DNA laddering. The effect of a nonlethal dose of HQ on the localization of apoptosis-induced factor (AIF) and phosphorylation of stress-activated kinase-2/p38 (SAPK2/p38) was detected by immunocytochemistry. Actin structures were visualized by phalloidin staining. Cell membrane blebbing was detected using GFP-membrane-labeled RPE cells (ARPE-GFP-c'-rRas). Changes in gene expression patterns of RPE cells within 48 hours of prolonged treatment with a nonlethal dose of HQ were evaluated by microarray analysis and confirmed by Northern blotting. RESULTS: The viability of RPE after a prolonged sublethal injury dose of HQ was determined by multiple assays and confirmed by the absence of AIF translocation or DNA laddering. Prolonged exposure (16 hours) of RPE cells to a nonlethal dose of HQ resulted in actin rearrangement into globular aggregates and cell membrane blebbing. Kinetic microarray analysis at several time points over a 48-hour recovery period revealed significant upregulation of genes involved in ameliorating the oxidative stress, chaperone proteins, anti-apoptotic factors, and DNA repair factors, and downregulation of pro-apoptotic genes. Genes involved in extracellular matrix functions were also dysregulated. Recovery of RPE cells after the injury was confirmed by the normalization of gene expression dysregulation back to baseline levels within 48 hours. CONCLUSIONS: RPE cells avoided cell death and recovered from prolonged oxidative injury by activating a host of defense mechanisms while simultaneously triggering genes and cellular responses that may be involved in RPE disease development.