RESUMO
Age-related macular degeneration (AMD) is associated with an overactive complement system and an increase in circulating antibodies. Our search for potential neoantigens that can trigger complement activation in disease has led us to investigate elastin. A loss of the elastin layer (EL) of Bruch's membrane (BrM) has been reported in aging and AMD together with an increase of serum elastin-derived peptides and α-elastin antibodies. In the mouse model of cigarette smoke exposure (CSE), damage in BrM, loss of the EL, and vision loss are dependent on complement activation. We have examined the hypothesis that CSE generates immunogenic elastin neoepitopes that trigger an increase in α-elastin IgG and IgM antibodies, which can then bind to the neoepitopes in the target cells or membranes, triggering complement activation. Specifically, we showed that immunization with elastin peptide oxidatively modified by cigarette smoke (ox-elastin) exacerbated ocular pathology and vision loss in CSE mice. In contrast, mice receiving peptide immunotherapy (PIT) with ox-elastin did not lose vision over the smoking period and exhibited a more preserved BrM. Immunization and PIT correlated with humoral immunity and complement activation and IgG/IgM deposition in the RPE/BrM/choroid. Finally, PIT modulated immune markers IFNγ and IL-4. The data further support the hypothesis that complement activation, triggered by immune complex formation in target tissues, plays a role in ocular damage in the CSE model. As PIT with ox-elastin peptides reduces damage, we discuss the possibility that AMD progression might be preventable.
Assuntos
Lâmina Basilar da Corioide , Degeneração Macular , Camundongos , Animais , Lâmina Basilar da Corioide/patologia , Elastina/metabolismo , Imunização , Degeneração Macular/metabolismo , Imunoglobulina M , Imunoglobulina GRESUMO
BACKGROUND: Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood-retina barrier of the immune privileged eye. METHODS: We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1ß and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. RESULTS: RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1ß to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. CONCLUSION: Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.
Assuntos
Degeneração Macular , Epitélio Pigmentado da Retina , Animais , Humanos , Camundongos , Ratos , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Inflamação/genética , Inflamação/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Pigmentos da Retina/genética , Pigmentos da Retina/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The extracellular matrix (ECM) and its turnover play a crucial role in the pathogenesis of several inflammatory diseases, including age-related macular degeneration (AMD). Elastin, a critical protein component of the ECM, not only provides structural and mechanical support to tissues, but also mediates several intracellular and extracellular molecular signaling pathways. Abnormal turnover of elastin has pathological implications. In the eye elastin is a major structural component of Bruch's membrane (BrM), a critical ECM structure separating the retinal pigment epithelium (RPE) from the choriocapillaris. Reduced integrity of macular BrM elastin, increased serum levels of elastin-derived peptides (EDPs), and elevated elastin antibodies have been reported in AMD. Existing reports suggest that elastases, the elastin-degrading enzymes secreted by RPE, infiltrating macrophages or neutrophils could be involved in BrM elastin degradation, thus contributing to AMD pathogenesis. EDPs derived from elastin degradation can increase inflammatory and angiogenic responses in tissues, and the elastin antibodies are shown to play roles in immune cell activity and complement activation. This review summarizes our current understanding on the elastases/elastin fragments-mediated mechanisms of AMD pathogenesis.
Assuntos
Elastina , Degeneração Macular , Lâmina Basilar da Corioide/patologia , Corioide/metabolismo , Humanos , Degeneração Macular/metabolismo , Peptídeos/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
PURPOSE: Age-related macular degeneration (AMD), the leading cause of blindness in western populations, is associated with an overactive complement system, and an increase in circulating antibodies against certain epitopes, including elastin. As loss of the elastin layer of Bruch's membrane (BrM) has been reported in aging and AMD, we previously showed that immunization with elastin peptide oxidatively modified by cigarette smoke (ox-elastin), exacerbated ocular pathology in the smoke-induced ocular pathology (SIOP) model. Here we asked whether ox-elastin peptide-based immunotherapy (PIT) ameliorates damage. METHODS: C57BL/6J mice were injected with ox-elastin peptide at two doses via weekly subcutaneous administration, while exposed to cigarette smoke for 6 months. FcγR-/- and uninjected C57BL/6J mice served as controls. Retinal morphology was assessed by electron microscopy, and complement activation, antibody deposition and mechanisms of immunological tolerance were assessed by Western blotting and ELISA. RESULTS: Elimination of Fcγ receptors, preventing antigen/antibody-dependent cytotoxicity, protected against SIOP. Mice receiving PIT with low dose ox-elastin (LD-PIT) exhibited reduced humoral immunity, reduced complement activation and IgG/IgM deposition in the RPE/choroid, and largely a preserved BrM. While there is no direct evidence of ox-elastin pathogenicity, LD-PIT reduced IFNγ and increased IL-4 within RPE/choroid. High dose PIT was not protective. CONCLUSIONS: These data further support ox-elastin role in ocular damage in part via elastin-specific antibodies, and support the corollary that PIT with ox-elastin attenuates ocular pathology. Overall, damage is associated with complement activation, antibody-dependent cell-mediated cytotoxicity, and altered cytokine signature.
Assuntos
Fumar Cigarros/efeitos adversos , Elastina/imunologia , Imunoterapia/métodos , Degeneração Macular/terapia , Peptídeos/uso terapêutico , Receptores de IgG/efeitos dos fármacos , Fumaça/efeitos adversos , Animais , Ativação do Complemento , Modelos Animais de Doenças , Elastina/metabolismo , Degeneração Macular/induzido quimicamente , Degeneração Macular/diagnóstico , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Peptídeos/imunologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestruturaRESUMO
PURPOSE: Age-related macular degeneration is a slowly progressing disease. Studies have tied disease risk to an overactive complement system. We have previously demonstrated that pathology in two mouse models, the choroidal neovascularization (CNV) model and the smoke-induced ocular pathology (SIOP) model, can be reduced by specifically inhibiting the alternative complement pathway (AP). Here we report on the development of a novel injury-site targeted inhibitor of the alternative pathway, and its characterization in models of retinal degeneration. METHODS: Expression of the danger associated molecular pattern, a modified annexin IV, in injured ARPE-19 cells was confirmed by immunohistochemistry and complementation assays using B4 IgM mAb. Subsequently, a construct was prepared consisting of B4 single chain antibody (scFv) linked to a fragment of the alternative pathway inhibitor, fH (B4-scFv-fH). ARPE-19 cells stably expressing B4-scFv-fH were microencapsulated and administered intravitreally or subcutaneously into C57BL/6 J mice, followed by CNV induction or smoke exposure. Progression of CNV was analyzed using optical coherence tomography, and SIOP using structure-function analyses. B4-scFv-fH targeting and AP specificity was assessed by Western blot and binding experiments. RESULTS: B4-scFv-fH was secreted from encapsulated RPE and inhibited complement in RPE monolayers. B4-scFv-fH capsules reduced CNV and SIOP, and western blotting for breakdown products of C3α, IgM and IgG confirmed a reduction in complement activation and antibody binding in RPE/choroid. CONCLUSIONS: Data supports a role for natural antibodies and neoepitope expression in ocular disease, and describes a novel strategy to target AP-specific complement inhibition to diseased tissue in the eye. PRECIS: AMD risk is tied to an overactive complement system, and ocular injury is reduced by alternative pathway (AP) inhibition in experimental models. We developed a novel inhibitor of the AP that targets an injury-specific danger associated molecular pattern, and characterized it in disease models.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Inativadores do Complemento/uso terapêutico , Via Alternativa do Complemento/efeitos dos fármacos , Modelos Animais de Doenças , Imunoglobulina M/imunologia , Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/metabolismo , Animais , Western Blotting , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Neovascularização de Coroide/diagnóstico por imagem , Neovascularização de Coroide/imunologia , Neovascularização de Coroide/terapia , Complemento C3/antagonistas & inibidores , Complemento C3/genética , Sistemas de Liberação de Medicamentos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão , Degeneração Retiniana/diagnóstico por imagem , Degeneração Retiniana/imunologia , Tomografia de Coerência Óptica , TransfecçãoRESUMO
To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5-/- zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5-/- mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.
Assuntos
Células Fotorreceptoras/patologia , Degeneração Retiniana , Epitélio Pigmentado da Retina/patologia , Proteínas de Transporte Vesicular/fisiologia , Transtornos da Visão/patologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transtornos da Visão/metabolismo , Peixe-ZebraRESUMO
The exocyst is a highly conserved protein complex found in most eukaryotic cells and is associated with many functions, including protein translocation in the endoplasmic reticulum, vesicular basolateral targeting, and ciliogenesis in the kidney. To investigate the exocyst functions, here we exchanged proline for alanine in the highly conserved VXPX ciliary targeting motif of EXOC5 (exocyst complex component 5), a central exocyst gene/protein, and generated stable EXOC5 ciliary targeting sequence-mutated (EXOC5CTS-m) Madin-Darby canine kidney (MDCK) cells. The EXOC5CTS-m protein was stable and could bind other members of the exocyst complex. Culturing stable control, EXOC5-overexpressing (OE), Exoc5-knockdown (KD), and EXOC5CTS-m MDCK cells on Transwell filters, we found that primary ciliogenesis is increased in EXOC5 OE cells and inhibited in Exoc5-KD and EXOC5CTS-m cells. Growing cells in collagen gels until the cyst stage, we noted that EXOC5-OE cells form mature cysts with single lumens more rapidly than control cysts, whereas Exoc5-KD and EXOC5CTS-m MDCK cells failed to form mature cysts. Adding hepatocyte growth factor to induce tubulogenesis, we observed that EXOC5-OE cell cysts form tubules more efficiently than control MDCK cell cysts, EXOC5CTS-m MDCK cell cysts form significantly fewer tubules than control cell cysts, and Exoc5-KD cysts did not undergo tubulogenesis. Finally, we show that EXOC5 mRNA almost completely rescues the ciliary phenotypes in exoc5-mutant zebrafish, unlike the EXOC5CTS-m mRNA, which could not efficiently rescue the phenotypes. Taken together, these results indicate that the exocyst, acting through the primary cilium, is necessary for renal ciliogenesis, cystogenesis, and tubulogenesis.
Assuntos
Cílios/fisiologia , Cistos/patologia , Túbulos Renais/crescimento & desenvolvimento , Rim/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , DNA Complementar/genética , Cães , Técnicas de Silenciamento de Genes , Humanos , Nefropatias/patologia , Células Madin Darby de Rim Canino , Mutagênese Sítio-Dirigida , Ligação Proteica , Transporte Proteico , RNA Mensageiro/metabolismo , Proteínas de Transporte Vesicular/genética , Peixe-ZebraRESUMO
Purpose: Risk for age-related macular degeneration (AMD), a slowly progressing, complex disease, is tied to an overactive complement system. Efforts are under way to develop an anticomplement-based treatment to be delivered locally or systemically. We developed an alternative pathway (AP) inhibitor fusion protein consisting of a complement receptor-2 fragment linked to the inhibitory domain of factor H (CR2-fH), which reduces the size of mouse choroidal neovascularization (CNV) when delivered locally or systemically. Specifically, we confirmed that ARPE-19 cells genetically engineered to produce CR2-fH reduce CNV lesion size when encapsulated and placed intravitreally. We extend this observation by delivering the encapsulated cells systemically in Matrigel. Methods: ARPE-19 cells were generated to stably express CR2 or CR2-fH, microencapsulated using sodium alginate, and injected subcutaneously in Matrigel into 2-month-old C57BL/6J mice. Four weeks after implantation, CNV was induced using argon laser photocoagulation. Progression of CNV was analyzed using optical coherence tomography. Bioavailability of CR2-fH was evaluated in Matrigel plugs with immunohistochemistry, as well as in ocular tissue with dot blots. Efficacy as an AP inhibitor was confirmed with protein chemistry. Results: An efficacious number of implanted capsules to reduce CNV was identified. Expression of the fusion protein systemically did not elicit an immune response. Bioavailability studies showed that CR2-fH was present in the RPE/choroid fractions of the treated mice, and reduced CNV-associated ocular complement activation. Conclusions: These findings indicate that systemic production of the AP inhibitor CR2-fH can reduce CNV in the mouse model.
Assuntos
Cápsulas/química , Encapsulamento de Células/métodos , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/terapia , Colágeno/química , Fator H do Complemento/antagonistas & inibidores , Inativadores do Complemento/farmacologia , Laminina/química , Proteoglicanas/química , Animais , Disponibilidade Biológica , Linhagem Celular , Fator H do Complemento/metabolismo , Inativadores do Complemento/metabolismo , Combinação de Medicamentos , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Domínios Proteicos , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Proteínas Recombinantes , Tomografia de Coerência ÓpticaRESUMO
Despite substantial opportunity and commercial interest in developing drugs that modulate the complement system in a broad range of non-orphan indications, several obstacles remain to be overcome. Among these issues is the biophysical nature of complement proteins, whose circulating levels are typically very high and whose turnover rates are relatively rapid, especially in the setting of chronic inflammatory conditions. This situation necessitates the use of very high levels of therapeutic compounds in order to achieve both multi-pathway and multiple effector mechanism inhibition. In addition, one must avoid infectious complications or the systemic impairment of the other important physiological functions of complement. Herein we focus on the development of a novel therapeutic strategy based on injured tissue-specific targeting of complement inhibitors using the antigen-combining domains of a small subset of natural IgM antibodies, which as endogenous antibodies specifically recognize sites of local damage across a broad range of tissues and locally activate complement C3, resulting in C3 fragment covalent fixation. Because the use of such recombinant tissue-targeting inhibitors precludes the utility of measuring systemic levels of complement biomarkers or function, since a goal of this targeting strategy is to leave those processes intact and unimpeded, we also briefly describe a new method designed to quantitatively measure using imaging modalities the inhibition of generation of fixed C3 fragments at sites of inflammation/injury. In addition to the ability to determine whether complement activation is locally constrained with the use of inhibitors, there is also a broader application of this imaging approach to inflammatory and autoimmune diseases characterized by local complement activation.
Assuntos
Inativadores do Complemento/uso terapêutico , Proteínas do Sistema Complemento/metabolismo , Inflamação/diagnóstico , Inflamação/terapia , Animais , Autoimunidade , Ativação do Complemento , Diagnóstico por Imagem , Humanos , Terapia de Alvo Molecular , Especificidade de ÓrgãosRESUMO
Purpose: Smoking and the incidence of age-related macular degeneration (AMD) have been linked to an overactive complement system. Here, we examined in a retrospective cohort study whether AMD-associated single nucleotide polymorphisms (SNPs), smoking, ethnicity, and disease status are correlated with blood complement levels. Methods: Population: The study involved 91 AMD patients and 133 controls, which included 73% Americans of European descent (EUR) and 27% Americans of African descent (AFR) in South Carolina. Readouts: Participants were genotyped for 10 SNPs and systemic levels of complement factor H (CFH) activity, and the complement activation products C3a, C5a, and Bb were assessed. Main Outcome Measures: Univariate and multivariable logistic regression models were used to examine associations between AMD status and distinct readouts. Results: AMD affects EUR individuals more than AFRs. EUR but not AFR AMD subjects revealed higher levels of Factors C3a and Bb. In all subjects, a 10-unit increase in C3a levels was associated with an approximately 10% increase in the odds of being AMD-positive, and C3a and Bb were associated with smoking. While CFH activity levels were not correlated with AMD, a significant interaction was evident between patient age and CFH activity. Finally, EURs had lower odds of AMD with enhanced copies of rs1536304 (VEGFA) and higher odds with more copy numbers of rs3766404 (CFH). Conclusions: Our results support previous studies of systemic complement components being potential biomarkers for AMD, but they suggest that smoking and disease do not synergistically affect complement levels. We also suggest a novel susceptibility and protective haplotypes in the South Carolinian AMD population. Our studies indicate that augmented complement activation associated with advanced AMD could be attributed to a decrease in CFH activity in younger patients.
Assuntos
Ativação do Complemento/genética , Fator H do Complemento/genética , Degeneração Macular/genética , Polimorfismo de Nucleotídeo Único , Fumar/genética , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , População Negra , Estudos de Casos e Controles , Complemento C3a/genética , Complemento C3a/imunologia , Complemento C5a/genética , Complemento C5a/imunologia , Fator B do Complemento/genética , Fator B do Complemento/imunologia , Fator H do Complemento/imunologia , Feminino , Expressão Gênica , Humanos , Modelos Logísticos , Degeneração Macular/etnologia , Degeneração Macular/imunologia , Degeneração Macular/patologia , Masculino , Estudos Retrospectivos , Fumar/etnologia , Fumar/imunologia , Fumar/fisiopatologia , South Carolina , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia , População BrancaRESUMO
Optimum cytosolic calcium concentrations support balanced mitochondrial respiration. However, cytosolic Ca2+ concentrations vary among cell types and excess Ca2+ can cause mitochondrial dysfunction. We optimized an isolation protocol to eliminate excess Ca2+ and thereby minimizing structural damage. Ca2+ uptake was monitored by measuring mitochondrial Ca2+-dependent PKA activity using cAMP ELISAs, and O2 consumption levels during mitochondrial respiration using high-resolution respirometry. 3â¯nM Ca2+ was found to increase cAMP levels and produce optimal state III respiration. Hence, optimized isolation of mitochondria from cell lines using calcium denudation provides the best platform for the study of Ca2+-dependent regulation of mitochondrial signaling.
Assuntos
Cálcio , Mitocôndrias/metabolismo , Cálcio/análise , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , AMP Cíclico/metabolismo , Citosol/química , Humanos , Oxigênio/metabolismoRESUMO
We previously have shown that the highly conserved eight-protein exocyst trafficking complex is required for ciliogenesis in kidney tubule cells. We hypothesized here that ciliogenic programs are conserved across organs and species. To determine whether renal primary ciliogenic programs are conserved in the eye, and to characterize the function and mechanisms by which the exocyst regulates eye development in zebrafish, we focused on exoc5, a central component of the exocyst complex, by analyzing both exoc5 zebrafish mutants, and photoreceptor-specific Exoc5 knock-out mice. Two separate exoc5 mutant zebrafish lines phenocopied exoc5 morphants and, strikingly, exhibited a virtual absence of photoreceptors, along with abnormal retinal development and cell death. Because the zebrafish mutant was a global knockout, we also observed defects in several ciliated organs, including the brain (hydrocephalus), heart (cardiac edema), and kidney (disordered and shorter cilia). exoc5 knockout increased phosphorylation of the regulatory protein Mob1, consistent with Hippo pathway activation. exoc5 mutant zebrafish rescue with human EXOC5 mRNA completely reversed the mutant phenotype. We accomplished photoreceptor-specific knockout of Exoc5 with our Exoc5 fl/fl mouse line crossed with a rhodopsin-Cre driver line. In Exoc5 photoreceptor-specific knock-out mice, the photoreceptor outer segment structure was severely impaired at 4 weeks of age, although a full-field electroretinogram indicated a visual response was still present. However, by 6 weeks, visual responses were eliminated. In summary, we show that ciliogenesis programs are conserved in the kidneys and eyes of zebrafish and mice and that the exocyst is necessary for photoreceptor ciliogenesis and retinal development, most likely by trafficking cilia and outer-segment proteins.
Assuntos
Cílios/metabolismo , Exocitose , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Células Fotorreceptoras de Vertebrados/patologia , Retina/patologia , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/metabolismo , Peixe-ZebraRESUMO
Retinal pigment epithelium (RPE) alterations in age-related macular degeneration occur in patches, potentially involving long-distance communication between damaged and healthy areas. Communication along the epithelium might be mediated by extracellular vesicles (EVs). To test this hypothesis, EVs were collected from supernatants of polarized ARPE-19 and primary porcine RPE monolayers for functional and biochemical assays. EVs from oxidatively stressed donor cells reduced barrier function in recipient RPE monolayers when compared to control EVs. The effect on barrier function was dependent on EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Mass spectrometry-based proteomic analysis of EVs identified HDAC6, which is known to reduce tight junction stability. Activity assays confirmed the presence of HDAC6 in EVs, and EV transfer assays using HDAC6 inhibitors confirmed its effect in monolayers. These findings demonstrate that EVs can communicate stress messages to healthy RPE cells, potentially contributing to RPE dysfunction.
Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , Desacetilase 6 de Histona/metabolismo , Degeneração Macular/metabolismo , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Animais , Linhagem Celular , Vesículas Extracelulares/patologia , Humanos , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/patologia , SuínosRESUMO
Age-related macular degeneration (AMD) is the leading cause of blindness in the USA. Polymorphisms in various complement components are associated with an increased risk for AMD, and it has been hypothesized that an overactive complement system is partially responsible for the pathology of AMD. AMD is classified as early, intermediate, or late AMD, depending on the degree of the associated pathologies. Late AMD can be characterized as either lesions associated with neovascular AMD or geographic atrophy. Both sets of lesions are associated with pathology at the RPE/choroid interface, which include a thickening of Bruch's membrane, presence of drusen, and pigmentary alterations, and deterioration of the blood-retina barrier has been reported. These changes can lead to the slow degeneration and atrophy of the photoreceptors in the macula in dry AMD, or progress to choroidal neovascularization (CNV) and leakage of these new vessels in wet AMD. It has been shown previously that complement anaphylatoxins C3a and C5a, signaling via their respective G-protein-coupled receptors, can alter RPE cell function and promote choroidal neovascularization. However, it is important to note these components also play a role in tissue repair. Here we discuss anaphylatoxin signaling in AMD-related target cells and the potential implications for the design of anti-complement therapeutics.
Assuntos
Ativação do Complemento , Complemento C3/imunologia , Complemento C5a/imunologia , Degeneração Macular/imunologia , Receptor da Anafilatoxina C5a/imunologia , Envelhecimento/imunologia , Animais , Lâmina Basilar da Corioide/patologia , Corioide/patologia , Neovascularização de Coroide/imunologia , Citocinas/biossíntese , Células Endoteliais/patologia , Fibrose , Previsões , Humanos , Inflamassomos/metabolismo , Inflamação , Degeneração Macular/tratamento farmacológico , Degeneração Macular/patologia , Camundongos , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/imunologia , Células Fotorreceptoras Retinianas Bastonetes/patologiaRESUMO
Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological or toxicological evaluation.
Assuntos
Inteligência Artificial , Imageamento Tridimensional/métodos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Animais , Carbonil Cianeto m-Clorofenil Hidrazona/análogos & derivados , Carbonil Cianeto m-Clorofenil Hidrazona/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Oxidantes/toxicidade , Fenótipo , ATPase Trocadora de Sódio-Potássio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , terc-Butil Hidroperóxido/metabolismoRESUMO
One of the affected tissues in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), a tissue that consists of terminally differentiated cells and that accumulates damage over time. In all tissues, mitochondria (mt), which play an essential role in both cell health (energy) and death (initiator of apoptosis), undergo an aging process through the accumulation of mtDNA damage, changes in mitochondrial dynamics, a reduction in biogenesis, and mitophagy, leading to an overall reduction in mitochondrial energy production and other non-energy-related functions. Here we have compared energy metabolism in primary human RPE cells isolated from aborted fetus or aged donor eyes and grown as stable monolayers. H2O2 treatment resulted in the generation of reactive oxygen species and superoxide, an effect that was significantly augmented by age. Mitochondrial metabolism, as analyzed by Seahorse respirometry, revealed reduced mitochondrial oxygen consumption (ATP production) at baseline and a complete loss of reserve capacity in aged cells. Likewise, glycolysis was blunted in aged cells. Taken together, these studies showed that RPE cells derived from aged donor eyes are more susceptible to oxidative stress, and exhibit a loss in mitochondrial respiratory reserve capacity and a reduction in glycolysis. These data suggest that while old cells may have sufficient energy at rest, they cannot mount a stress response requiring additional ATP and reducing agents. In summary, these data support the hypothesis that mitochondria or energy metabolism is a valid target for therapy in AMD.
Assuntos
Envelhecimento/metabolismo , Metabolismo Energético , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Idoso , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxidantes/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Cultura Primária de Células , Ionóforos de Próton/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/embriologia , Superóxidos/metabolismoRESUMO
Millions of individuals older than 50-years suffer from age-related macular degeneration (AMD). Associated with this multifactorial disease are polymorphisms of complement factor genes and a main environmental risk factor-oxidative stress. Until now the linkage between these risk factors for AMD has not been fully understood. Recent studies, integrating results on oxidative stress, complement activation, epidemiology and ocular pathology suggested the following sequence in AMD-etiology: initially, chronic oxidative stress results in modification of proteins and lipids in the posterior of the eye; these tissue alterations trigger chronic inflammation, involving the complement system; and finally, invasive immune cells facilitate pathology in the retina. Here, we summarize the results for animal studies which aim to elucidate this molecular interplay of oxidative events and tissue-specific complement activation in the eye.
Assuntos
Ativação do Complemento , Proteínas do Sistema Complemento/metabolismo , Degeneração Macular/metabolismo , Estresse Oxidativo , Animais , Proteínas do Sistema Complemento/genética , Modelos Animais de Doenças , Humanos , Degeneração Macular/genética , Camundongos Knockout , Retina/metabolismo , Retina/patologiaRESUMO
One feature common to many of the pathways implicated in retinal degeneration is increased metabolic stress leading to impaired mitochondrial function. We found that exposure of cells to calcium ionophores or oxidants as metabolic stressors diminish maximal mitochondrial capacity. A library of 50,000 structurally diverse "drug-like" molecules was screened for protection against loss of calcium-induced loss of mitochondrial capacity in 661W rod-derived cells and C6 glioblastomas. Initial protective hits were then tested for protection against IBMX-induced loss of mitochondrial capacity as measured via respirometry. Molecules that protected mitochondria were then evaluated for protection of rod photoreceptor cells in retinal explants from rd1 mice. Two of the molecules attenuated loss of photoreceptor cells in the rd1 model. In the 661W cells, exposure to calcium ionophore or tert-butylhydroperoxide caused mitochondrial fragmentation that was blocked with the both compounds. Our studies have identified molecules that protect mitochondria and attenuate loss of photoreceptors in models of retinal degeneration suggesting that they could be good leads for development of therapeutic drugs for treatment of a wide variety of retinal dystrophies.
Assuntos
Mitocôndrias/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Retinose Pigmentar/prevenção & controle , Bibliotecas de Moléculas Pequenas/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Glicólise/efeitos dos fármacos , Camundongos Mutantes , Mitocôndrias/metabolismo , Técnicas de Cultura de Órgãos , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Retinose Pigmentar/genética , Retinose Pigmentar/metabolismoRESUMO
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.