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1.
Adv Exp Med Biol ; 1415: 67-71, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37440016

RESUMEN

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.


Asunto(s)
Lámina Basal de la Coroides , Degeneración Macular , Ratones , Animales , Lámina Basal de la Coroides/patología , Elastina/metabolismo , Inmunización , Degeneración Macular/metabolismo , Inmunoglobulina M , Inmunoglobulina G
2.
Transl Vis Sci Technol ; 12(7): 17, 2023 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-37462980

RESUMEN

Purpose: Risk for developing age-related macular degeneration (AMD) is linked to an overactive complement system. In the mouse model of laser-induced choroidal neovascularization (CNV), elevated levels of complement effector molecules, including complement C3, have been identified, and the alternative pathway (AP) is required for pathology. The main soluble AP regular is complement factor H (fH). We have previously shown that AP inhibition via subretinal AAV-mediated delivery of CR2-fH using a constitutive promoter is efficacious in reducing CNV. Here we ask whether the C3 promoter (pC3) effectively drives CR2-fH bioavailability for gene therapy. Methods: Truncated pC3 was used to generate plasmids pC3-mCherry/CR2-fH followed by production of corresponding AAV5 vectors. pC3 activation was determined in transiently transfected ARPE-19 cells stimulated with H2O2 or normal human serum (+/- antioxidant or humanized CR2-fH, respectively). CNV was analyzed in C57BL/6J mice treated subretinally with AAV5-pC3-mCherry/CR2-fH using imaging (optical coherence tomography [OCT] and fundus imaging), functional (electroretinography [ERG]), and molecular (protein expression) readouts. Results: Modulation of pC3 in vitro is complement and oxidative stress dependent, as shown by mCherry fluorescence. AAV5-pC3-CR2-fH were identified as safe and effective using OCT and ERG. CR2-fH expression significantly reduced CNV compared to mCherry and was correlated with reduced levels of C3dg/C3d in the retinal pigment epithelium/choroid fraction. Conclusions: We conclude that complement-dependent regulation of AP inhibition ameliorates AMD pathology as effectively as using a constitutive promoter. Translational Relevance: The goal of anticomplement therapy is to restore homeostatic levels of complement activation, which might be more easily achievable using a self-regulating system.


Asunto(s)
Neovascularización Coroidal , Degeneración Macular Húmeda , Ratones , Animales , Humanos , Vía Alternativa del Complemento/genética , Peróxido de Hidrógeno/farmacología , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Neovascularización Coroidal/genética , Neovascularización Coroidal/terapia , Degeneración Macular Húmeda/genética , Degeneración Macular Húmeda/terapia
3.
Exp Eye Res ; 212: 108755, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34487725

RESUMEN

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.


Asunto(s)
Fumar Cigarrillos/efectos adversos , Elastina/inmunología , Inmunoterapia/métodos , Degeneración Macular/terapia , Péptidos/uso terapéutico , Receptores de IgG/efectos de los fármacos , Humo/efectos adversos , Animales , Activación de Complemento , Modelos Animales de Enfermedad , Elastina/metabolismo , Degeneración Macular/inducido químicamente , Degeneración Macular/diagnóstico , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Péptidos/inmunología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura
4.
Invest Ophthalmol Vis Sci ; 62(4): 11, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33830174

RESUMEN

Purpose: The risk for age-related macular degeneration has been tied to an overactive complement system. Despite combined attempts by academia and industry to develop therapeutics that modulate the complement response, particularly in the late geographic atrophy form of advanced AMD, to date, there is no effective treatment. We have previously demonstrated that pathology in the smoke-induced ocular pathology (SIOP) model, a model with similarities to dry AMD, is dependent on activation of the alternative complement pathway and that a novel complement activation site targeted inhibitor of the alternative pathway can be delivered to ocular tissues via an adeno-associated virus (AAV). Methods: Two different viral vectors for specific tissue targeting were compared: AAV5-VMD2-CR2-fH for delivery to the retinal pigment epithelium (RPE) and AAV2YF-smCBA-CR2-fH for delivery to retinal ganglion cells (RGCs). Efficacy was tested in SIOP (6 months of passive smoke inhalation), assessing visual function (optokinetic responses), retinal structure (optical coherence tomography), and integrity of the RPE and Bruch's membrane (electron microscopy). Protein chemistry was used to assess complement activation, CR2-fH tissue distribution, and CR2-fH transport across the RPE. Results: RPE- but not RGC-mediated secretion of CR2-fH was found to reduce SIOP and complement activation in RPE/choroid. Bioavailability of CR2-fH in RPE/choroid could be confirmed only after AAV5-VMD2-CR2-fH treatment, and inefficient, adenosine triphosphate-dependent transport of CR2-fH across the RPE was identified. Conclusions: Our results suggest that complement inhibition for AMD-like pathology is required basal to the RPE and argues in favor of AAV vector delivery to the RPE or outside the blood-retina barrier.


Asunto(s)
Activación de Complemento/efectos de los fármacos , Inactivadores del Complemento/administración & dosificación , Degeneración Macular/tratamiento farmacológico , Epitelio Pigmentado de la Retina/patología , Animales , Coroides , Modelos Animales de Enfermedad , Inyecciones Intravítreas , Degeneración Macular/metabolismo , Degeneración Macular/patología , Ratones , Ratones Endogámicos C57BL , Retina , Epitelio Pigmentado de la Retina/efectos de los fármacos , Tomografía de Coherencia Óptica
5.
Exp Eye Res ; 207: 108583, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33878326

RESUMEN

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.


Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inactivadores del Complemento/uso terapéutico , Vía Alternativa del Complemento/efectos de los fármacos , Modelos Animales de Enfermedad , Inmunoglobulina M/inmunología , Degeneración Retiniana/terapia , Epitelio Pigmentado de la Retina/metabolismo , Animales , Western Blotting , Línea Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Neovascularización Coroidal/diagnóstico por imagen , Neovascularización Coroidal/inmunología , Neovascularización Coroidal/terapia , Complemento C3/antagonistas & inhibidores , Complemento C3/genética , Sistemas de Liberación de Medicamentos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión , Degeneración Retiniana/diagnóstico por imagen , Degeneración Retiniana/inmunología , Tomografía de Coherencia Óptica , Transfección
6.
Mol Vis ; 26: 370-377, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32476817

RESUMEN

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.


Asunto(s)
Cápsulas/química , Encapsulación Celular/métodos , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/terapia , Colágeno/química , Factor H de Complemento/antagonistas & inhibidores , Inactivadores del Complemento/farmacología , Laminina/química , Proteoglicanos/química , Animales , Disponibilidad Biológica , Línea Celular , Factor H de Complemento/metabolismo , Inactivadores del Complemento/metabolismo , Combinación de Medicamentos , Expresión Génica , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Dominios Proteicos , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Proteínas Recombinantes , Tomografía de Coherencia Óptica
7.
J Vis Exp ; (157)2020 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-32281978

RESUMEN

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.


Asunto(s)
Productos Biológicos/química , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Sistemas de Liberación de Medicamentos/métodos , Ojo/efectos de los fármacos , Animales , Cápsulas , Modelos Animales de Enfermedad , Ratones
8.
Invest Ophthalmol Vis Sci ; 61(3): 45, 2020 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-32207814

RESUMEN

Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness in Western populations. While an overactive complement system has been linked to pathogenesis, mechanisms contributing to its activation are largely unknown. In aged and AMD eyes, loss of the elastin layer (EL) of Bruch's membrane (BrM) has been reported. Elastin antibodies are elevated in patients with AMD, the pathogenic significance of which is unclear. Here we assess the role of elastin antibodies using a mouse model of smoke-induced ocular pathology (SIOP), which similarly demonstrates EL loss. Methods: C57BL/6J mice were immunized with elastin or elastin peptide oxidatively modified by cigarette smoke (ox-elastin). Mice were then exposed to cigarette smoke or air for 6 months. Visual function was assessed by optokinetic response, retinal morphology by spectral-domain optical coherence tomography and electron microscopy, and complement activation and antibody deposition by Western blot. Results: Ox-elastin IgG and IgM antibodies were elevated in ox-elastin immunized mice following 6 months of smoke, whereas elastin immunization had a smaller effect. Ox-elastin immunization exacerbated smoke-induced vision loss, with thicker BrM and more damaged retinal pigment epithelium (RPE) mitochondria compared with mice immunized with elastin or nonimmunized controls. These changes were correlated with increased levels of IgM, IgG2, IgG3, and complement activation products in RPE/choroid. Conclusions: These data demonstrate that SIOP mice generate elastin-specific antibodies and that immunization with ox-elastin exacerbates ocular pathology. Elastin antibodies represented complement fixing isotypes that, together with the increased presence of complement activation seen in immunized mice, suggest that elastin antibodies exert pathogenic effects through mediating complement activation.


Asunto(s)
Autoanticuerpos/sangre , Lámina Basal de la Coroides/patología , Modelos Animales de Enfermedad , Elastina/inmunología , Atrofia Geográfica/etiología , Epitelio Pigmentado de la Retina/patología , Fumar/efectos adversos , Animales , Western Blotting , Activación de Complemento/fisiología , Proteínas del Sistema Complemento/metabolismo , Sensibilidad de Contraste/fisiología , Ensayo de Inmunoadsorción Enzimática , Atrofia Geográfica/inmunología , Atrofia Geográfica/patología , Inmunización , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Ratones , Ratones Endogámicos C57BL , Nistagmo Optoquinético/fisiología , Oxidación-Reducción , Productos de Tabaco , Agudeza Visual/fisiología
9.
Mol Immunol ; 108: 8-12, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30763805

RESUMEN

Age-related macular degeneration (AMD) is the leading cause of blindness in the US. Polymorphisms in complement components are associated with increased AMD risk, and it has been hypothesized that an overactive complement system is partially responsible for AMD pathology. Choroidal neovascularization (CNV) has two phases, injury/angiogenesis and repair/fibrosis. Complement activation has been shown to be involved in the angiogenesis phase of murine CNV, but has not been investigated during repair. Anaphylatoxin (C3a and C5a) signaling in particular has been shown to be involved in both tissue injury and repair in other models. CNV was triggered by laser-induced photocoagulation in C57BL/6 J mice, and lesion sizes measured by optical coherence tomography. Alternative pathway (AP) activation or C3a-receptor (C3aR) and C5a-receptor (C5aR) engagement was inhibited during the repair phase only of CNV with the AP-inhibitor CR2-fH, a C3aR antagonist (N2-[(2,2-diphenylethoxy)acetyl]-l-arginine, TFA), or a C5a blocking antibody (CLS026), respectively. Repair after CNV was also investigated in C3aR/C5aR double knockout mice. CR2-fH treatment normalized anaphylatoxin levels in the eye and accelerated regression of CNV lesions. In contrast, blockade of anaphylatoxin-receptor signaling pharmacologically or genetically did not significantly alter the course of lesion repair. These results suggest that continued complement activation prevents fibrotic scar resolution, and emphasizes the importance of reducing anaphylatoxins to homeostatic levels. This duality of complement, playing a role in injury and repair, will need to be considered when selecting a complement inhibitory strategy for AMD.


Asunto(s)
Neovascularización Coroidal/inmunología , Vía Alternativa del Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Receptor de Anafilatoxina C5a/inmunología , Receptores de Complemento/inmunología , Regeneración/inmunología , Animales , Neovascularización Coroidal/genética , Neovascularización Coroidal/patología , Vía Alternativa del Complemento/genética , Proteínas del Sistema Complemento/genética , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Receptor de Anafilatoxina C5a/genética , Receptores de Complemento/genética , Regeneración/genética
10.
Transl Vis Sci Technol ; 7(2): 3, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29576927

RESUMEN

PURPOSE: Age-related macular degeneration (AMD) is a slowly progressing disease, and risk appears to be tied to an overactive complement system. We have previously demonstrated that mouse choroidal neovascularization (CNV) and smoke-induced ocular pathology can be reduced with an alternative pathway (AP) inhibitor fusion protein consisting of a complement receptor-2 fragment linked to the inhibitory domain of factor H (CR2-fH) when delivered systemically. Here we developed an experimental approach with genetically engineered encapsulated ARPE-19 cells to produce CR2-fH intravitreally. METHODS: ARPE-19 cells were generated to stably express CR2 or CR2-fH, microencapsulated using sodium alginate, and injected intravitreally into 2-month-old C57BL/6J mice. CNV was induced using argon laser photocoagulation 4 weeks postinjection. Presence of capsules and progression of CNV was analyzed using optical coherence tomography. Bioavailability of CR2-fH was evaluated in retina sections by immunohistochemistry, and efficacy as an AP inhibitor by C3a ELISA. RESULTS: Secretion of CR2-fH or CR2 from encapsulated ARPE-19 cells was confirmed. An efficacious concentration of CR2-fH capsules to reduce CNV was identified. Bioavailability studies showed that CR2-fH was present in capsules and retinas of injected mice, and reduced CNV-associated ocular C3a production. CONCLUSIONS: These findings indicate that the AP inhibitor CR2-fH, when generated intravitreally, can reduce CNV in mouse. TRANSLATIONAL RELEVANCE: Encapsulated ARPE-19 cells secreting CR2-fH or perhaps other antiangiogenic or prosurvival factors might be useful as a potential therapeutic tool to treat age-related macular degeneration.

11.
Mol Ther Methods Clin Dev ; 9: 1-11, 2018 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-29234687

RESUMEN

Complement activation plays a significant role in age-related macular degeneration (AMD) pathogenesis, and polymorphisms interfering with factor H (fH) function, a complement alternative pathway (AP) inhibitor, are associated with increased AMD risk. We have previously validated an AP inhibitor, a fusion protein consisting of a complement receptor 2 fragment linked to the inhibitory domain of fH (CR2-fH) as an efficacious treatment for choroidal neovascularization (CNV) when delivered intravenously. Here we tested an alternative approach of AAV-mediated delivery (AAV5-VMD2-CR2-fH or AAV5-VMD2-mCherry) using subretinal delivery in C57BL/6J mice. Secretion of CR2-fH was confirmed in polarized retinal pigment epithelium (RPE) cells. A safe concentration of AAV5-VMD2-CR2-fH was identified using electroretinography, optical coherence tomography (OCT), RPE morphology, and antibody profiling. One month after gene delivery, CNV was induced using argon laser photocoagulation. OCT assessment demonstrated reduced CNV with AAV5-VMD2-CR2-fH administration. Bioavailability studies revealed that gene-therapy delivered similar levels of CR2-fH to the RPE/choroid as treatment by intravenous injections, and C3a ELISA verified reduced CNV-associated ocular C3a production. These results contribute to existing data illustrating the importance of the AP of complement in CNV development and its potential role in AMD treatment. Demonstration of AAV-vector efficacy opens new avenues for the development of treatment strategies.

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