RESUMEN
Neovascular age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). An overactive complement system is associated with AMD pathogenesis, and serum pro-inflammatory cytokines, including IL-17, are elevated in AMD patients. IL-17 is produced by complement C5a-receptor-expressing T-cells. In murine CNV, infiltrating γδT- rather than Th17-cells produce the IL-17 measurable in lesioned eyes. Here we asked whether C5a generated locally in response to CNV recruits IL-17-producing T-cells to the eye. CNV lesions were generated using laser photocoagulation and quantified by imaging; T-lymphocytes were characterized by QRT-PCR. CNV resulted in an increase in splenic IL-17-producing γδT- and Th17-cells; yet in the CNV eye, only elevated levels of γδT-cells were observed. Systemic administration of anti-C5- or anti-C5a-blocking antibodies blunted the CNV-induced production of splenic Th17- and γδT-cells, reduced CNV size and eliminated ocular γδT-cell infiltration. In ARPE-19 cell monolayers, IL-17 triggered a pro-inflammatory state; and splenocyte proliferation was elevated in response to ocular proteins. Thus, we demonstrated that CNV lesions trigger a systemic immune response, augmenting local ocular inflammation via the infiltration of IL-17-producing γδT-cells, which are presumably recruited to the eye in a C5a-dependent manner. Understanding the complexity of complement-mediated pathological mechanisms will aid in the development of an AMD treatment.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Coroides/inmunología , Neovascularización Coroidal/inmunología , Complemento C5a/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Células Th17/inmunología , Inmunidad Adaptativa , Animales , Anticuerpos Neutralizantes/farmacología , Linfocitos T CD8-positivos/patología , Línea Celular , Movimiento Celular/efectos de los fármacos , Coroides/patología , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/etiología , Neovascularización Coroidal/genética , Complemento C5a/antagonistas & inhibidores , Expresión Génica , Humanos , Inmunidad Innata , Inyecciones Intravenosas , Interleucina-17/genética , Interleucina-17/inmunología , Fotocoagulación/efectos adversos , Ratones , Ratones Endogámicos C57BL , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/inmunología , Bazo/inmunología , Bazo/patología , Células Th17/patologíaRESUMEN
PURPOSE: Loss of CD46 has recently been implicated in choroidal neovascularization in mice. Herein we investigated the effect of nitrite modification of the extracellular matrix (ECM) as an in vitro model of "aging" and its effect on CD46 expression and vascular endothelial growth factor (VEGF) release in cocultured human retinal pigment epithelium (RPE). METHODS: ARPE-19 cells were plated onto RPE-derived ECM conditions (untreated; nitrite modified; nitrite modified followed by washing with Triton X-100; or nitrite modified followed by washing with Triton X-100 and coated with extracellular matrix ligands). Cells were cultured for 7 days and CD46 expression was analyzed by immunohistochemistry and Western blot. Additionally, CD46 short interfering RNA (siRNA) was transfected into ARPE-19 cells, and VEGF levels were determined by ELISA. Finally, in the same ECM conditions, ARPE-19 cells were challenged with normal human serum and VEGF levels determined by ELISA. RESULTS: CD46 is expressed on the basolateral surface of ARPE-19 cells on RPE-derived ECM. Nitrite modification of ECM reduced the expression of CD46 on ARPE-19 cells by 0.5-fold (P = 0.003) and increased VEGF release in ARPE-19 cells by 1.7-fold (P < 0.001). CD46 knockdown also increased release of VEGF on the apical and basal sides of ARPE-19 cells in culture by 1.3- (P = 0.012) and 1.2-fold (P = 0.017), respectively. CONCLUSIONS: Nitrite modification of the ECM decreased CD46 expression and increased the release of VEGF from ARPE-19 cells. Changes in CD46 expression may lead to changes in VEGF and play a pathologic role in the development of age-related macular degeneration.
Asunto(s)
Neovascularización Coroidal/genética , ADN/genética , Regulación de la Expresión Génica , Proteína Cofactora de Membrana/genética , Nitritos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Western Blotting , Células Cultivadas , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Humanos , Proteína Cofactora de Membrana/biosíntesis , Ratones , Ratones Noqueados , Microscopía Confocal , Epitelio Pigmentado de la Retina/patologíaRESUMEN
PURPOSE: Complement factor B (CFB) is a required component of the alternative pathway (AP) of complement, and CFB polymorphisms are associated with age-related macular degeneration (AMD) risk. Complement factor B is made in the liver, but expression has also been detected in retina and retinal pigment epithelium (RPE)-choroid. We investigated whether production of CFB by the RPE can promote AP activation in mouse choroidal neovascularization (CNV). METHODS: Transgenic mice expressing CFB under the RPE65 promoter were generated and crossed onto factor B-deficient (CFB-KO) mice. Biological activity was determined in vitro using RPE monolayers and in vivo using laser-induced CNV. Contribution of systemic CFB was investigated using CFB-KO reconstituted with CFB-sufficient serum. RESULTS: Transgenic mice (CFB-tg) expressed CFB in RPE-choroid; no CFB was detected in serum. Cultured CFB-tg RPE monolayers secreted CFB apically and basally upon exposure to oxidative stress that was biologically active. Choroidal neovascularization sizes were comparable between wild-type and CFB-tg mice, but significantly increased when compared to lesions in CFB-KO mice. Injections of CFB-sufficient serum into CFB-KO mice resulted in partial reconstitution of systemic AP activity and significantly increased CNV size. CONCLUSIONS: Mouse RPE cells express and secrete CFB sufficient to promote RPE damage and CNV. This further supports that local complement production may regulate disease processes; however, the reconstitution experiments suggest that additional components may be sequestered from the bloodstream. Understanding the process of ocular complement production and regulation will further our understanding of the AMD disease process and the requirements of a complement-based therapeutic.
Asunto(s)
Coroides/patología , Neovascularización Coroidal/genética , Factor B del Complemento/genética , Vía Alternativa del Complemento/genética , Regulación de la Expresión Génica , ARN Mensajero/genética , Animales , Western Blotting , Células Cultivadas , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Factor B del Complemento/biosíntesis , Modelos Animales de Enfermedad , Electrorretinografía , Ensayo de Inmunoadsorción Enzimática , Rayos Láser/efectos adversos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/patología , Epitelio Pigmentado de la Retina/fisiopatología , Tomografía de Coherencia ÓpticaRESUMEN
Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the elderly. To study potential underlying mechanisms of AMD, animal models are utilized, focusing mostly on mice. Recently, genomic and phenotypic differences between the so-called control substrains, C57BL/6J and C57BL/6N, have been described in models of ocular and non-ocular diseases. In particular, the rd8 mutation of the Crb1 gene present in the C57BL/6N has been shown to impact certain ocular phenotypes and appears to augment phenotypes generally associated with inflammation. Here, we investigated angiogenic factor and cytokine expression using pathway arrays as well as the susceptibility to laser-induced choroidal neovascularization (CNV), a model of wet AMD, in the two substrains. Age-matched 3-month-old C57BL/6J and C57BL/6N animals differed in gene expression levels for angiogenic factors and cytokines, with 6N animals expressing higher levels of inflammatory markers than 6Js. Yet laser-induced CNV was comparable in size between the two substrains. This lack of difference in CNV size was correlated with a gene expression profile that was comparable between the two substrains, due to the fact that the degree of change in gene expression of inflammatory markers after CNV was blunted in 6N mice. In summary, significant gene expression differences exist between C57BL/6J and C57BL/6N animals, reinforcing the notion that appropriate litter-mate controls or genetic background controls need to be used. Contrary to our expectation, CNV was not augmented in 6N animals, suggesting that low chronic inflammation in the RPE might provide a level of pre-conditioning and protection against stress.
Asunto(s)
Quimiocinas/genética , Neovascularización Coroidal/genética , Regulación de la Expresión Génica , ARN/genética , Animales , Quimiocinas/biosíntesis , Neovascularización Coroidal/etiología , Neovascularización Coroidal/metabolismo , Citocinas/biosíntesis , Citocinas/genética , Modelos Animales de Enfermedad , Femenino , Rayos Láser/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , FenotipoRESUMEN
Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD.
Asunto(s)
Activación de Complemento/fisiología , Estrés del Retículo Endoplásmico/fisiología , Lípidos/análisis , Estrés Oxidativo/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Humo , Acetilcisteína/farmacología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Compuestos de Bencidrilo/farmacología , Western Blotting , Células Cultivadas , Activación de Complemento/efectos de los fármacos , Factor B del Complemento/genética , Factor B del Complemento/metabolismo , Vía Alternativa del Complemento/efectos de los fármacos , Vía Alternativa del Complemento/fisiología , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Depuradores de Radicales Libres/farmacología , Proteínas de Choque Térmico/genética , Humanos , Metabolismo de los Lípidos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nicotina/farmacología , Estrés Oxidativo/efectos de los fármacos , Receptores de Complemento/antagonistas & inhibidores , Receptores de Complemento/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nicotiana/química , Factor de Transcripción CHOP/genéticaRESUMEN
Age-related macular degeneration (AMD) is a slowly progressing multifactorial disease involving genetic abnormalities and environmental insults. Genetic studies have demonstrated that polymorphisms in different complement proteins increase the risk for developing AMD. Previously, we have shown that in retinal pigment epithelium (RPE) monolayers, exposure to oxidative stress reduced complement inhibition on the cell surface, with the resulting increase in complement activation leading to vascular endothelial growth factor (VEGF) release and VEGF-receptor-2-mediated disruption of the monolayer barrier function. Complement activation was found to be sublytic and transient and require the assembly of the membrane attack complex (MAC). Here, we asked how this transient, sublytic complement activation could trigger long-term pathological changes in RPE cells. The initial activation of the L-type voltage-gated calcium channels was followed by calcium influx and activation of several kinases. While Erk/Ras activation was found to be transient, Src kinase phosphorylation was sustained. We have shown previously that Src kinase controls VEGF release from RPE cells by altering the activity of the L-type channel. We propose that the prolonged Src kinase activation, and its resulting effects on membrane depolarization and calcium influx, leads to sustained VEGF secretion. In addition, the previously shown effect of the autocrine positive feedback loop in RPE cells, involving VEGF-induced VEGF production and secretion via VEGFR-2 receptors, will augment and prolong the effects of sublytic complement activation. In summary, identification of the links between oxidative stress, chronic, low-grade activation of the complement system, and elevated VEGF expression and secretion might offer opportunities to selectively inhibit pathological VEGF release only.
Asunto(s)
Activación de Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/patología , Familia-src Quinasas/metabolismo , Canales de Calcio Tipo L/fisiología , Células Cultivadas , Proteínas del Sistema Complemento/inmunología , Activación Enzimática/inmunología , Humanos , Degeneración Macular/inmunología , Degeneración Macular/metabolismo , Estrés Oxidativo/inmunología , Epitelio Pigmentado de la Retina/enzimología , Epitelio Pigmentado de la Retina/inmunología , Familia-src Quinasas/inmunologíaRESUMEN
BACKGROUND: Age-related macular degeneration (AMD), a complex disease involving genetic variants and environmental insults, is among the leading causes of blindness in Western populations. Genetic and histologic evidence implicate the complement system in AMD pathogenesis; and smoking is the major environmental risk factor associated with increased disease risk. Although previous studies have demonstrated that cigarette smoke exposure (CE) causes retinal pigment epithelium (RPE) defects in mice, and smoking leads to complement activation in patients, it is unknown whether complement activation is causative in the development of CE pathology; and if so, which complement pathway is required. METHODS: Mice were exposed to cigarette smoke or clean, filtered air for 6 months. The effects of CE were analyzed in wildtype (WT) mice or mice without a functional complement alternative pathway (AP; CFB(-/-) ) using molecular, histological, electrophysiological, and behavioral outcomes. RESULTS: CE in WT mice exhibited a significant reduction in function of both rods and cones as determined by electroretinography and contrast sensitivity measurements, concomitant with a thinning of the nuclear layers as measured by SD-OCT imaging and histology. Gene expression analyses suggested that alterations in both photoreceptors and RPE/choroid might contribute to the observed loss of function, and visualization of complement C3d deposition implies the RPE/Bruch's membrane (BrM) complex as the target of AP activity. RPE/BrM alterations include an increase in mitochondrial size concomitant with an apical shift in mitochondrial distribution within the RPE and a thickening of BrM. CFB(-/-) mice were protected from developing these CE-mediated alterations. CONCLUSIONS: Taken together, these findings provide clear evidence that ocular pathology generated in CE mice is dependent on complement activation and requires the AP. Identifying animal models with RPE/BrM damage and verifying which aspects of pathology are dependent upon complement activation is essential for developing novel complement-based treatment approaches for the treatment of AMD.
Asunto(s)
Proteínas del Sistema Complemento/metabolismo , Oftalmopatías/patología , Oftalmopatías/fisiopatología , Transducción de Señal/efectos de los fármacos , Humo/efectos adversos , Animales , Complemento C3/metabolismo , Proteínas del Sistema Complemento/deficiencia , Proteínas del Sistema Complemento/genética , Oftalmopatías/inducido químicamente , Oftalmopatías/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Lectinas/metabolismo , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Factores de Tiempo , Nicotiana/efectos adversosRESUMEN
Uncontrolled activation of the alternative complement pathway (AP) is thought to be associated with age-related macular degeneration. Previously, we have shown that in retinal pigmented epithelial (RPE) monolayers, oxidative stress reduced complement inhibition on the cell surface, resulting in sublytic complement activation and loss of transepithelial resistance (TER), but the potential ligand and pathway involved are unknown. ARPE-19 cells were grown as monolayers on transwell plates, and sublytic complement activation was induced with H2O2 and normal human serum. TER deteriorated rapidly in H2O2-exposed monolayers upon adding normal human serum. Although the effect required AP activation, AP was not sufficient, because elimination of MASP, but not C1q, prevented TER reduction. Reconstitution experiments to unravel essential components of the lectin pathway (LP) showed that both ficolin and mannan-binding lectin can activate the LP through natural IgM antibodies (IgM-C2) that recognize phospholipid cell surface modifications on oxidatively stressed RPE cells. The same epitopes were found on human primary embryonic RPE monolayers. Likewise, mouse laser-induced choroidal neovascularization, an injury that involves LP activation, could be increased in antibody-deficient rag1(-/-) mice using the phospholipid-specific IgM-C2. In summary, using a combination of depletion and reconstitution strategies, we have shown that the LP is required to initiate the complement cascade following natural antibody recognition of neoepitopes, which is then further amplified by the AP. LP activation is triggered by IgM bound to phospholipids. Taken together, we have defined novel mechanisms of complement activation in oxidatively stressed RPE, linking molecular events involved in age-related macular degeneration, including the presence of natural antibodies and neoepitopes.
Asunto(s)
Complemento C1q/metabolismo , Vía Alternativa del Complemento , Lectina de Unión a Manosa de la Vía del Complemento , Inmunoglobulina M/sangre , Estrés Oxidativo , Fosfolípidos/sangre , Epitelio Pigmentado de la Retina/metabolismo , Animales , Línea Celular , Complemento C1q/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Ratones , Ratones Noqueados , Oxidantes/farmacología , Epitelio Pigmentado de la Retina/patologíaAsunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/inmunología , Degeneración Macular/inmunología , Epitelio Pigmentado de la Retina/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas del Sistema Complemento/inmunología , Feto/citología , Humanos , Degeneración Macular/metabolismo , Degeneración Macular/patología , Estrés Oxidativo/inmunología , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Uncontrolled activation of the alternative complement pathway and secretion of vascular endothelial growth factor (VEGF) are thought to be associated with age-related macular degeneration (AMD). Previously, we have shown that in RPE monolayers, oxidative-stress reduced complement inhibition on the cell surface. The resulting increased level of sublytic complement activation resulted in VEGF release, which disrupted the barrier facility of these cells as determined by transepithelial resistance (TER) measurements. Induced rather than basal VEGF release in RPE is thought to be controlled by different mechanisms, including voltage-dependent calcium channel (VDCC) activation and mitogen-activated protein kinases. Here we examined the potential intracellular links between sublytic complement activation and VEGF release in RPE cells challenged with H(2)O(2) and complement-sufficient normal human serum (NHS). Disruption of barrier function by H(2)O(2) + NHS rapidly increased Ras expression and Erk and Src phosphorylation, but had no effect on P38 phosphorylation. Either treatment alone had little effect. TER reduction could be attenuated by inhibiting Ras, Erk and Src activation, or blocking VDCC or VEGF-R2 activation, but not by inhibiting P38. Combinatorial analysis of inhibitor effects demonstrated that sublytic complement activation triggers VEGF secretion via two pathways, Src and Ras-Erk, with the latter being amplified by VEGF-R2 activation, but has no effect on constitutive VEGF secretion mediated via P38. Finally, effects on TER were directly correlated with release of VEGF; and sublytic MAC activation decreased levels of zfp36, a negative modulator of VEGF transcription, resulting in increased VEGF expression. Taken together, identifying how sublytic MAC induces VEGF expression and secretion might offer opportunities to selectively inhibit pathological VEGF release only.
Asunto(s)
Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Canales de Calcio/metabolismo , Línea Celular , Activación de Complemento/efectos de los fármacos , Activación de Complemento/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Peróxido de Hidrógeno/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Oxidantes/farmacología , Epitelio Pigmentado de la Retina/citología , Tristetraprolina/biosíntesis , Proteínas ras/biosíntesis , Familia-src Quinasas/biosíntesisRESUMEN
PURPOSE: Autophagy is a lysosomal machinery-dependent process that catabolizes cellular components/organelles and proteins in an autophagic vacuole (AV)-dependent and -independent manner, respectively. Short-term exposure of the retina to bright light results in shortening of the outer segments, concomitant with AV formation. Autophagy is also induced by continuous long-term light damage, leading to photoreceptor cell death. Here the authors examined two questions: is autophagy induced during light damage proapoptotic or antiapoptotic, and are rods and cones affected differently? To this end, Balb/c mice exposed to light damage were treated with rapamycin to increase autophagy. METHODS: Balb/c and GFP-LC3 mice were treated with rapamycin/vehicle. Photoreceptor degeneration was induced by 10-day light damage. Autophagy was documented by histologic, biochemical, and molecular tools; rod and cone survival was assessed by histology and electroretinography. RESULTS: Light damage resulted in rod, but not cone, cell loss. Autophagy and AV formation was elicited in response to light damage, which was amplified by rapamycin. Rapamycin treatment significantly improved rod survival and function, reduced apoptosis, and normalized cytokine production that was increased in light damage. However, AV formation in GFP-LC3 mice revealed that light damage or rapamycin treatment induced AVs in cones, concomitant with reduced cone-mediated electroretinograms. CONCLUSIONS: Systemic rapamycin treatment provided rod protection; however, AV formation was induced only in cones. Thus, rapamycin may act differentially in stressed photoreceptors; rapamycin might protect rods by normalizing cytokine production, removing damaged proteins by AV-independent autophagy, or both, whereas cones might be protected by AV-dependent autophagy, possibly involving reduced photon capture.
Asunto(s)
Inmunosupresores/farmacología , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Degeneración Retiniana/tratamiento farmacológico , Células Fotorreceptoras Retinianas Bastones/efectos de los fármacos , Sirolimus/farmacología , Animales , Apoptosis/fisiología , Autofagia/fisiología , Western Blotting , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Electrorretinografía , Femenino , Inyecciones Intraperitoneales , Luz/efectos adversos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Traumatismos Experimentales por Radiación/patología , Degeneración Retiniana/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Human genetic studies have demonstrated that polymorphisms in different complement proteins can increase the risk for developing AMD. There are three pathways of complement activation, classical (CP), alternative (AP), and lectin (LP), which all activate a final common pathway. Proteins encoded by the AMD risk genes participate in the AP (CFB), CP/LP (C2), or in the AP and final common pathway (C3). Here we tested which pathway is essential in mouse laser-induced CNV. CNV was analyzed using single complement pathway knockouts (i.e., eliminating one complement pathway at a time), followed by a double knockout in which only the AP is present, and the CP and LP are disabled, using molecular, histological and electrophysiological outcomes. First, single-gene knockouts were analyzed and compared to wild type mice; C1q(-/-) (no CP), MBL(-/-) (no LP), and CFB(-/-) (no AP). Six days after the laser-induced lesion, mice without a functional AP had reduced CNV progression (P<0.001) and preserved ERG amplitudes, whereas those without a functional CP or LP were indistinguishable from the wild type controls (P>0.3). Second, AP-only mice (C1q(-/-)MBL(-/-)) were as protected from developing CNV as the CFB(-/-) mice. The degree of pathology in each strain correlated with protein levels of the angiogenic and anti-angiogenic protein VEGF and PEDF, respectively, as well as levels of terminal pathway activation product C5a, and C9. The analysis of complement activation pathways in mouse laser-induced CNV allows for the following conclusions. Comparing the single pathway knockouts with those having only a functional AP showed: (1) that AP activation is necessary, but not alone sufficient for injury; and (2) that initial complement activation proceeds via both the LP and CP. Thus, these data indicate an important role for the AP in the generation of complement-dependent injury in the RPE and choroid via amplification of CP- and LP-initiated complement activation. Improving our understanding of the local regulation of this pathway in the eye is essential for developing improved treatment approaches for AMD.
Asunto(s)
Neovascularización Coroidal/inmunología , Neovascularización Coroidal/patología , Vía Alternativa del Complemento/inmunología , Rayos Láser , Retina/inmunología , Retina/patología , Inductores de la Angiogénesis/metabolismo , Animales , Neovascularización Coroidal/fisiopatología , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Electrorretinografía , Regulación de la Expresión Génica , Ratones , Células Fotorreceptoras de Vertebrados/inmunología , Células Fotorreceptoras de Vertebrados/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Genetic variations in complement factor H (fH), an inhibitor of the complement alternative pathway (CAP), and oxidative stress are associated with age-related macular degeneration (AMD). Recently, novel complement therapeutics have been created with the capacity to be "targeted" to sites of complement activation. One example is our recombinant form of fH, CR2-fH, which consists of the N-terminus of mouse fH that contains the CAP-inhibitory domain, linked to a complement receptor 2 (CR2) targeting fragment that binds complement activation products. CR2-fH was investigated in vivo in the mouse model of choroidal neovascularization (CNV) and in vitro in oxidatively stressed RPE cell monolayers. RPE deterioration and CNV development were found to require CAP activation, and specific CAP inhibition by CR2-fH reduced the loss of RPE integrity and angiogenesis in CNV. In both the in vivo and in vitro paradigm of RPE damage, a model requiring molecular events known to be involved in AMD, complement-dependent VEGF production, was confirmed. These data may open new avenues for AMD treatment strategies.
Asunto(s)
Neovascularización Coroidal/tratamiento farmacológico , Inactivadores del Complemento/farmacología , Vía Alternativa del Complemento/efectos de los fármacos , Degeneración Macular/tratamiento farmacológico , Epitelio Pigmentado de la Retina/efectos de los fármacos , Animales , Línea Celular , Neovascularización Coroidal/inmunología , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Humanos , Técnicas In Vitro , Degeneración Macular/inmunología , Degeneración Macular/patología , Ratones , Modelos Biológicos , Estrés Oxidativo , Epitelio Pigmentado de la Retina/inmunología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
PURPOSE: In a previous study, several quantitative trait loci (QTL) that influence age-related degeneration (ageRD) were identified in a cross between the albino strains B6(Cg)-Tyr(c-2J)/J (B6a) and BALB/cByJ (C). The Chromosome (Chr) 6 and Chr 10 QTL were the strongest and most highly significant loci and both involved B6a protective alleles. The QTL were responsible for 21% and 9% of the variance in phenotypes, respectively. We focused on these two QTL to identify candidate genes. METHODS: DNA microarrays were used for the two mouse strains at four and eight months of age to identify genes that are differentially regulated and map to either QTL. Gene Ontology (GO) analysis of the differentially expressed genes was performed to identify possible processes and pathways associated with ageRD. To identify additional candidates, database analyses (Positional Medline or PosMed) were used. Based on differential expression, PosMed, and the presence of reported polymorphisms, five genes per QTL were selected for further study by sequencing analysis and qRT-PCR. Tumor necrosis factor, alpha- induced protein 3 (Tnfaip3; on a C57BL/6J (B6) background) was phenotypically tested. Single nucleotide polymorphisms (SNPs) flanking this gene were correlated with outer nuclear layer thickness (ONL), and eight-month-old Tnfaip3(+/-) mice were tested for ageRD. RESULTS: Polymorphisms were found in the coding regions of eight genes. Changes in gene expression were identified by qRT-PCR for Hexokinase 2 (Hk2) and Docking protein 1 (Dok1) at four months and for Dok1 and Tnfaip3 at eight months. Tnfaip3 was selected for phenotypic testing due to differential expression and the presence of two nonsynonymous mutations. However, when ONL thickness was compared in eight-month-old congenic Tnfaip3(+/-) and Tnfaip3(+/+) mice, no differences were found, suggesting that Tnfaip3 is not the quantitative trait gene (QTG) for the Chr 10 QTL. The GO analysis revealed that GO terms associated with stress and cell remodeling are overrepresented in the ageRD-sensitive C strain compared with the B6a strain with age (eight months). In the ageRD-resistant B6a strain, compared with the C strain, GO terms associated with antioxidant response and the regulation of blood vessel size are overrepresented with age. CONCLUSIONS: The analyses of differentially expressed genes and the PosMed database yielded candidate genes for the Chr 6 and Chr 10 QTL. HtrA serine peptidase 2 (Htra2), Dok1, and Tnfaip3 were deemed most promising because of their known roles in apoptosis and our finding of nonsynonymous substitutions between B6a and C strains. While Tnfaip3 was excluded as the QTG for the Chr 10 QTL, Dok1 and Htra2 remain good candidates for the Chr 6 QTL. Finally, the GO term analysis further supports the general hypothesis that oxidative stress is involved in ageRD.
Asunto(s)
Mapeo Cromosómico , Estudios de Asociación Genética , Sitios de Carácter Cuantitativo , Degeneración Retiniana/genética , Animales , Cisteína Endopeptidasas/genética , Proteínas de Unión al ADN/genética , Femenino , Serina Peptidasa A2 que Requiere Temperaturas Altas , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos , Ratones Noqueados , Análisis por Micromatrices , Proteínas Mitocondriales/genética , Estrés Oxidativo , Fenotipo , Fosfoproteínas/genética , Polimorfismo de Nucleótido Simple , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
We have recently shown that oxidative stress of ARPE-19 cells alters the expression of the cell surface complement regulatory proteins DAF and CD59, and permits increased activation of complement when the cells are subsequently exposed to serum. Based upon these results, we hypothesized that RPE cells respond to cellular stress as if it is infection, and reduce their surface expression of complement regulatory proteins to foster the local immune response. To test this hypothesis, we examined whether cellular hypoxia would produce a similar change in ARPE-19 cells. In addition, we asked whether this response to oxidative stress is universal in all epithelial cells, by examining the expression of complement regulatory proteins on the surface of the renal and pulmonary epithelial cells. We found that the expression of complement regulatory proteins is altered by aseptic cellular stressors such as hypoxia and oxidative stress, but the response to these conditions differs from tissue to tissue. In RPE cells oxidative stress reduces the expression of the cell surface complement regulators and sensitizes the cells to complement mediated injury. This specific response is not seen in epithelial cells from the lung or kidney, and is not induced by hypoxia. These studies help explain the unique mechanisms by which uncontrolled complement activation may contribute to the development of AMD.
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Membrana Celular/inmunología , Proteínas del Sistema Complemento/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Animales , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Hipoxia de la Célula/inmunología , Línea Celular , Activación de Complemento/inmunología , Citometría de Flujo , Humanos , Túbulos Renales/patología , Pulmón/patología , Proteína Cofactora de Membrana/metabolismo , Ratones , Especificidad de Órganos/inmunología , Estrés Oxidativo/inmunología , Receptores de Complemento/metabolismo , Receptores de Complemento 3bRESUMEN
PURPOSE: RPE65, a major retinal pigment epithelium protein, is essential in generating 11-cis retinal, the chromophore for all opsins. Without chromophore, cone opsins are mislocalized and cones degenerate rapidly (e.g., Rpe65(-/-) mouse). Function, survival, and correct targeting of opsins is increased in Rpe65(-/-) cones on supplying 11-cis retinal. Here, we determine the consequences of 11-cis retinal withdrawal and supplementation on cone development in the all-cone Nrl(-/-) retina. METHODS: Rpe65(-/-) Nrl(-/-), Nrl(-/-), and wild-type mice were examined. Cone structure was analyzed by using TUNEL assay, electron microscopy, and cone-specific antibodies. Cone function was assessed with light-adapted single-flash ERGs. RESULTS: Rpe65(-/-)Nrl(-/-) mice had an increased number of TUNEL-positive photoreceptors during programmed cell death compared with Nrl(-/-) mice, in addition to accelerated age-related degeneration. Cone function in Rpe65(-/-)Nrl(-/-) mice was minimal, and opsins were mislocalized. Treatment with 11-cis retinal restored cone function, promoted outer segment formation, and enabled opsin trafficking to outer segments. Eliminating Rpe65 prevented rosette formation in Nrl(-/-) retinas; supplementation of Rpe65(-/-)Nrl(-/-) mice with 11-cis retinal resulted in their reoccurrence. CONCLUSIONS: Taken together, function and opsin trafficking in Nrl(-/-) and wild-type cones are comparable, confirming and extending our findings that cone maturation and outer segment development are dependent on the presence of chromophore. The data on age-related cone death in Rpe65(-/-)Nrl(-/-) mice and the reintroduction of rosettes after 11-cis retinal injections confirm that outer segments, which for steric reasons appear to introduce rosettes in an all-cone retina, are essential for cell survival. These results are important for understanding and treating chromophore-related cone dystrophies.
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Apoptosis , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Proteínas Portadoras/fisiología , Proteínas del Ojo/fisiología , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Conos/fisiología , Degeneración Retiniana/fisiopatología , Retinaldehído/fisiología , Animales , Electrorretinografía , Técnica del Anticuerpo Fluorescente Indirecta , Etiquetado Corte-Fin in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Microscopía Fluorescente , Opsinas/metabolismo , Estimulación Luminosa , Degeneración Retiniana/metabolismo , Retinaldehído/administración & dosificación , cis-trans-IsomerasasRESUMEN
Uncontrolled activation of the alternative pathway of complement is thought to be associated with age-related macular degeneration (AMD). The alternative pathway is continuously activated in the fluid phase, and tissue surfaces require continuous complement inhibition to prevent spontaneous autologous tissue injury. Here, we examined the effects of oxidative stress on the ability of immortalized human retinal pigment epithelial cells (ARPE-19) to regulate complement activation on their cell surface. Combined treatment with H(2)O(2) (to induce oxidative stress) and complement-sufficient serum was found to disrupt the barrier function of stable ARPE-19 monolayers as determined by transepithelial resistance (TER) measurements. Neither treatment alone had any effect. TER reduction was correlated with increased cell surface deposition of C3, and could be prevented by using C7-depleted serum, an essential component of the terminal complement pathway. Treatment with H(2)O(2) reduced surface expression of the complement inhibitors DAF, CD55, and CD59, and impaired regulation at the cell surface by factor H present within the serum. Combined treatment of the monolayers with H(2)O(2) and serum elicited polarized secretion of vascular epidermal growth factor (VEGF). Both, secretion of VEGF and TER reduction could be attenuated using either an alternative pathway inhibitor or by blocking VEGF receptor-1/2 signaling. Regarded together, these studies demonstrate that oxidative stress reduces regulation of complement on the surface of ARPE-19 cells, increasing complement activation. This sublytic activation results in VEGF release, which mediates disruption of the cell monolayer. These findings link oxidative stress, complement activation, and apical VEGF release, which have all been associated with the pathogenesis of AMD.
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Proteínas del Sistema Complemento/metabolismo , Epitelio Pigmentado de la Retina/lesiones , Epitelio Pigmentado de la Retina/metabolismo , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Línea Celular , Factor H de Complemento/metabolismo , Vía Alternativa del Complemento , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Humanos , Degeneración Macular/etiología , Degeneración Macular/inmunología , Degeneración Macular/metabolismo , Estrés Oxidativo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
PURPOSE: Polymorphisms in factor H (fH), an inhibitor of the alternative pathway (AP) of complement activation, are associated with increased risk for age-related macular degeneration (AMD). The authors investigated the therapeutic use of a novel recombinant form of fH, CR2-fH, which is targeted to sites of complement activation, in mouse choroidal neovascularization (CNV). CR2-fH consists of the N terminus of mouse fH, which contains the AP-inhibitory domain, linked to a complement receptor 2 (CR2) targeting fragment that binds complement activation products. METHODS: Laser-induced CNV was analyzed in factor-B-deficient mice or in mice treated with CR2-fH, soluble CR2 (targeting domain), or PBS. CNV progression was analyzed by molecular, histologic, and electrophysiological readouts. RESULTS: Intravenously administered CR2-fH reduced CNV size, preserved retina function, and abrogated the injury-associated expression of C3 and VEGF mRNA. CR2 and PBS treatment was without effect. In therapeutically relevant paradigms involving delayed treatment after injury, CR2-fH was effective in reducing CNV and provided approximately 60% of the amount of protection of that seen in factor B-deficient mice that lacked functional AP. After intravenous injection, CR2-fH localized to sites of C3 deposition in RPE-choroid. CONCLUSIONS: Specific inhibition of the AP reduces angiogenesis in mouse CNV. Of note, intravenous injection of C3d-targeted CR2-fH is protective even though endogenous fH is present in serum at a higher relative concentration, and serum fH contains native C3d and cell surface binding domains that target it to cell surfaces. The most common AMD-associated variant of fH resides within a native cell-binding region of fH (Tyr402His). These data may open new avenues for AMD treatment strategies.
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Neovascularización Coroidal/tratamiento farmacológico , Vía Alternativa del Complemento/efectos de los fármacos , Modelos Animales de Enfermedad , Degeneración Macular/tratamiento farmacológico , Proteínas Recombinantes de Fusión/administración & dosificación , Animales , Coroides/metabolismo , Neovascularización Coroidal/fisiopatología , Activación de Complemento/efectos de los fármacos , Complemento C3/antagonistas & inhibidores , Complemento C3/genética , Factor H de Complemento/antagonistas & inhibidores , Vía Alternativa del Complemento/inmunología , Electrorretinografía , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Infusiones Intravenosas , Degeneración Macular/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Retina/fisiopatología , Epitelio Pigmentado de la Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
PURPOSE: Genetic risk factors such as variations in complement factors H (CFH) and B (CFB) have been implicated in the etiology of age-related macular degeneration. It has been hypothesized that inadequate control of complement-driven inflammation may be a major factor in disease pathogenesis. The authors tested the involvement of the complement system in an experimental model for oxidative stress-mediated photoreceptor degeneration, the light-damage mouse model. METHODS: Changes in gene expression were assessed in BALB/c retinas in response to constant-light (CL) exposure using microarrays and real-time PCR. Susceptibility to CL exposure was tested in CFD(-/-) mice on a BALB/c background. Eyes were analyzed using electrophysiologic and histologic techniques. RESULTS: Genes encoding for proteins involved in complement activation were significantly upregulated after CL. The altered gene profiles were similar to proteins accumulated in drusen and to genes identified in the retina and RPE/choroid of patients with age-related macular degeneration. Cyclic-light reared CFD(-/-) and CFD(+/+) mice had indistinguishable rod function and number; however, after CL challenge, CFD(-/-) photoreceptors were significantly protected. CONCLUSIONS: These results suggest that rod degeneration in the CL-damaged retina involves the activity of the alternative complement pathway and that eliminating the alternative pathway is neuroprotective. Thus, the light damage albino mouse model may be a good model to study complement-mediated photoreceptor degeneration.
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Luz , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Traumatismos Experimentales por Radiación/prevención & control , Degeneración Retiniana/prevención & control , Animales , Activación de Complemento/genética , Factor D del Complemento/fisiología , Vía Alternativa del Complemento/fisiología , Electrorretinografía , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Traumatismos Experimentales por Radiación/genética , Traumatismos Experimentales por Radiación/patología , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Studies on human and animal models of retinal dystrophy have suggested that apoptosis may be the common pathway of photoreceptor cell death. Autophagy, the major cellular degradation process in animal cells, is important in normal development and tissue remodeling, as well as under pathological conditions. Previously we provided evidence that genes, whose products are involved in apoptosis and autophagy, may be coexpressed in photoreceptors undergoing degeneration. Here, we investigated autophagy in oxidative stress-mediated cell death in photoreceptors, analyzing the light-damage mouse model and 661W photoreceptor cells challenged with H(2)O(2). In the in vivo model, we demonstrated a time-dependent increase in the number of TUNEL-positive cells, concomitant with the formation of autophagosomes. In vitro, oxidative stress increased mRNA levels of apoptotic and autophagic marker genes. H(2)O(2) treatment resulted in the accumulation of TUNEL-positive cells, the majority of which contain autophagosomes. To determine whether autophagy and apoptosis might precede each other or co-occur, we performed inhibitor studies. The autophagy inhibitor 3-methyladenine (3-MA), silencing RNA (siRNA) against two genes whose products are required for autophagy (autophagy-related (ATG) gene 5 and beclin 1), as well as the pan-caspase-3 inhibitor, Zvad-fmk, were both found to partially block cell death. Blocking autophagy also significantly decreased caspase-3 activity, whereas blocking apoptosis increased the formation of autophagosomes. The survival effects of 3?MA and zVAD-fmk were not additive; rather treatment with both inhibitors lead to increased cell death by necrosis. In summary, the study first suggests that autophagy participates in photoreceptor cell death possibly by initiating apoptosis. Second, it confirms that cells that normally die by apoptosis will execute cell death by necrosis if the normal pathway is blocked. And third, these results argue that the up-stream regulators of autophagy need to be identified as potential therapeutic targets in photoreceptor degeneration.