RESUMO
Our previous studies have shown that systemic neonatal murine cytomegalovirus (MCMV) infection of BALB/c mice spread to the eye with subsequent establishment of latency in choroid/RPE. In this study, RNA sequencing (RNA-Seq) analysis was used to determine the molecular genetic changes and pathways affected by ocular MCMV latency. MCMV (50 pfu per mouse) or medium as control were injected intra-peritoneally (i.p.) into BALB/c mice at <3 days after birth. At 18 months post injection, the mice were euthanized, and the eyes were collected and prepared for RNA-Seq. Compared to three uninfected control eyes, we identified 321 differentially expressed genes (DEGs) in six infected eyes. Using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA), we identified 17 affected canonical pathways, 10 of which function in neuroretinal signaling, with the majority of DEGs being downregulated, while 7 pathways function in upregulated immune/inflammatory responses. Retinal and epithelial cell death pathways involving both apoptosis and necroptosis were also activated. MCMV ocular latency is associated with upregulation of immune and inflammatory responses and downregulation of multiple neuroretinal signaling pathways. Cell death signaling pathways are also activated and contribute to the degeneration of photoreceptors, RPE, and choroidal capillaries.
Assuntos
Infecções por Citomegalovirus , Infecções Oculares Virais , Muromegalovirus , Camundongos , Animais , Camundongos Endogâmicos BALB C , Infecções Oculares Virais/metabolismo , Infecções Oculares Virais/patologia , Corioide/metabolismo , Muromegalovirus/fisiologia , Perfilação da Expressão GênicaRESUMO
Although pathologies associated with acute virus infections have been extensively studied, the effects of long-term latent virus infections are less well understood. Human cytomegalovirus, which infects 50% to 80% of humans, is usually acquired during early life and persists in a latent state for the lifetime. The purpose of this study was to determine whether systemic murine cytomegalovirus (MCMV) infection acquired early in life disseminates to and becomes latent in the eye and if ocular MCMV can trigger in situ inflammation and occurrence of ocular pathology. This study found that neonatal infection of BALB/c mice with MCMV resulted in dissemination of virus to the eye, where it localized principally to choroidal endothelia and pericytes and less frequently to the retinal pigment epithelium (RPE) cells. MCMV underwent ocular latency, which was associated with expression of multiple virus genes and from which MCMV could be reactivated by immunosuppression. Latent ocular infection was associated with significant up-regulation of several inflammatory/angiogenic factors. Retinal and choroidal pathologies developed in a progressive manner, with deposits appearing at both basal and apical aspects of the RPE, RPE/choroidal atrophy, photoreceptor degeneration, and neovascularization. The pathologies induced by long-term ocular MCMV latency share features of previously described human ocular diseases, such as age-related macular degeneration.
Assuntos
Envelhecimento/patologia , Corioide/patologia , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Muromegalovirus/fisiologia , Retina/patologia , Indutores da Angiogênese/metabolismo , Animais , Animais Recém-Nascidos , Antígenos Virais/metabolismo , Corioide/diagnóstico por imagem , Corioide/ultraestrutura , Corioide/virologia , DNA Viral/metabolismo , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/diagnóstico por imagem , Interações Hospedeiro-Patógeno , Terapia de Imunossupressão , Inflamação/patologia , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Fagócitos/patologia , Retina/diagnóstico por imagem , Retina/ultraestrutura , Retina/virologia , Epitélio Pigmentado da Retina/diagnóstico por imagem , Epitélio Pigmentado da Retina/patologia , Tomografia de Coerência Óptica , Ativação ViralRESUMO
Age-related macular degeneration (AMD) is a complex, multifactorial, progressive disease which represents a leading cause of irreversible visual impairment and blindness in older individuals. Human cytomegalovirus (HCMV), which infects 50-80% of humans, is usually acquired during early life and persists in a latent state for the life of the individual. In view of its previously described pro-angiogenic properties, we hypothesized that cytomegalovirus might be a novel risk factor for progression to an advanced form, neovascular AMD, which is characterized by choroidal neovascularization (CNV). The purpose of this study was to investigate if latent ocular murine cytomegalovirus (MCMV) infection exacerbated the development of CNV in vascular endothelial growth factor (VEGF)-overexpressing VEGF-Ahyper mice. Here we show that neonatal infection with MCMV resulted in dissemination of virus to various organs throughout the body including the eye, where it localized principally to the choroid in both VEGF-overexpressingVEGF-Ahyper and wild-type(WT) 129 mice. By 6 months post-infection, no replicating virus was detected in eyes and extraocular tissues, although virus DNA was still present in all eyes and extraocular tissues of both VEGF-Ahyper and WT mice. Expression of MCMV immediate early (IE) 1 mRNA was detected only in latently infected eyes of VEGF-Ahyper mice, but not in eyes of WT mice. Significantly increased CNV was observed in eyes of MCMV-infected VEGF-Ahyper mice compared to eyes of uninfected VEGF-Ahyper mice, while no CNV lesions were observed in eyes of either infected or uninfected WT mice. Protein levels of several inflammatory/angiogenic factors, particularly VEGF and IL-6, were significantly higher in eyes of MCMV-infected VEGF-Ahyper mice, compared to uninfected controls. Initial studies of ocular tissue from human cadavers revealed that HCMV DNA was present in four choroid/retinal pigment epithelium samples from 24 cadavers. Taken together, our data suggest that ocular HCMV latency could be a significant risk factor for the development of AMD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Neovascularização de Coroide/virologia , Retinite por Citomegalovirus/virologia , Degeneração Macular/virologia , Muromegalovirus/patogenicidade , Retina/virologia , Latência Viral , Idoso , Idoso de 80 Anos ou mais , Animais , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Retinite por Citomegalovirus/genética , Retinite por Citomegalovirus/metabolismo , Retinite por Citomegalovirus/patologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Proteínas Imediatamente Precoces/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Transgênicos , Pessoa de Meia-Idade , Retina/metabolismo , Retina/ultraestrutura , Fatores de Risco , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The neovascular (wet) form of age-related macular degeneration (AMD) leads to vision loss due to choroidal neovascularization (CNV). Since macrophages are important in CNV development, and cytomegalovirus (CMV)-specific IgG serum titers in patients with wet AMD are elevated, we hypothesized that chronic CMV infection contributes to wet AMD, possibly by pro-angiogenic macrophage activation. This hypothesis was tested using an established mouse model of experimental CNV. At 6 days, 6 weeks, or 12 weeks after infection with murine CMV (MCMV), laser-induced CNV was performed, and CNV severity was determined 4 weeks later by analysis of choroidal flatmounts. Although all MCMV-infected mice exhibited more severe CNV when compared with control mice, the most severe CNV developed in mice with chronic infection, a time when MCMV-specific gene sequences could not be detected within choroidal tissues. Splenic macrophages collected from mice with chronic MCMV infection, however, expressed significantly greater levels of TNF-α, COX-2, MMP-9, and, most significantly, VEGF transcripts by quantitative RT-PCR assay when compared to splenic macrophages from control mice. Direct MCMV infection of monolayers of IC-21 mouse macrophages confirmed significant stimulation of VEGF mRNA and VEGF protein as determined by quantitative RT-PCR assay, ELISA, and immunostaining. Stimulation of VEGF production in vivo and in vitro was sensitive to the antiviral ganciclovir. These studies suggest that chronic CMV infection may serve as a heretofore unrecognized risk factor in the pathogenesis of wet AMD. One mechanism by which chronic CMV infection might promote increased CNV severity is via stimulation of macrophages to make pro-angiogenic factors (VEGF), an outcome that requires active virus replication.
Assuntos
Corioide/irrigação sanguínea , Neovascularização de Coroide/etiologia , Infecções por Herpesviridae/imunologia , Ativação de Macrófagos , Muromegalovirus/imunologia , Animais , Corioide/patologia , Neovascularização de Coroide/metabolismo , Doença Crônica , Modelos Animais de Doenças , Feminino , Infecções por Herpesviridae/complicações , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
To report an unusual case of endogenous fungal endophthalmitis due to Candida dubliniensis. Interventional case report of a 27-year-old immunocompetent male with loss of vision, dense vitritis, and chorioretinal infiltrates, who underwent a diagnostic pars plana vitrectomy. Microbiology cultures obtained by a diagnostic vitrectomy were positive for the growth of C. dubliniensis. This infectious process was then appropriately treated with intravitreal amphotericin B and systemic fluconazole with resolution of the endophthalmitis. Endogenous fungal endophthalmitis is a condition that can masquerade other more common causes of endophthalmitis. Atypical cases of endophthalmitis may benefit from diagnostic pars plana vitrectomy for prompt diagnosis and treatment.
Assuntos
Candida/isolamento & purificação , Endoftalmite/microbiologia , Infecções Oculares Fúngicas/microbiologia , Corpo Vítreo/microbiologia , Adulto , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Diagnóstico Diferencial , Endoftalmite/patologia , Endoftalmite/terapia , Infecções Oculares Fúngicas/patologia , Infecções Oculares Fúngicas/terapia , Humanos , Injeções Intravítreas , Masculino , Vitrectomia , Corpo Vítreo/patologia , Corpo Vítreo/cirurgia , West VirginiaRESUMO
Epidemiological data suggest that estrogen deficiency in postmenopausal women may contribute to the severity of AMD. We discovered that 17beta-estradiol (E2) was a crucial regulator of the severity of extracellular matrix turnover (ECM) dysregulation both in vivo and in vitro. We also found in vitro that the presence of estrogen receptor (ER)beta regulates MMP-2 activity. Therefore in an attempt to delineate the role of the ER subtypes, female estrogen receptor knockout (ERKO) mice were fed a high-fat diet, and the eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light over 2 weeks. Three months after cessation of blue light treatment, transmission electron microscopy was performed to assess severity of deposits, Bruchs membrane changes, and choriocapillaris endothelial morphology. We found that changes in the trimolecular complex of pro-MMP-2, MMP-14 and TIMP-2 correlated with increased Bruch's membrane thickening or sub-retinal deposit formation (basal laminar deposits) in ERKObeta mice. In addition RPE isolated from ERKObeta mice had an increase in expression of total collagen and a decrease in MMP-2 activity. Finally we found that ERK an intermediate signaling molecule in the MMP pathway was activated in RPE isolated from ERKObeta mice. These data suggest that mice which lack ERbeta are more susceptible to in vivo injury associated with environmental light and high fat diet.
Assuntos
Receptor beta de Estrogênio/fisiologia , Matriz Extracelular/metabolismo , Degeneração Macular/prevenção & controle , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Animais , Western Blotting , Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/ultraestrutura , Técnicas de Cultura de Células , Colágeno/metabolismo , Gorduras na Dieta/administração & dosagem , Matriz Extracelular/ultraestrutura , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Hibridização In Situ , Luz , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Epitélio Pigmentado da Retina/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
Recent epidemiological studies link Periodontal disease(PD) to age-related macular degeneration (AMD). We documented earlier that Porphyromonas gingivalis(Pg), keystone oral-pathobiont, causative of PD, efficiently invades human gingival epithelial and blood-dendritic cells. Here, we investigated the ability of dysbiotic Pg-strains to invade human-retinal pigment epithelial cells(ARPE-19), their survival, intracellular localization, and the pathological effects, as dysfunction of RPEs leads to AMD. We show that live, but not heat-killed Pg-strains adhere to and invade ARPEs. This involves early adhesion to ARPE cell membrane, internalization and localization of Pg within single-membrane vacuoles or cytosol, with some nuclear localization apparent. No degradation of Pg or localization inside double-membrane autophagosomes was evident, with dividing Pg suggesting a metabolically active state during invasion. We found significant downregulation of autophagy-related genes particularly, autophagosome complex. Antibiotic protection-based recovery assay further confirmed distinct processes of adhesion, invasion and amplification of Pg within ARPE cells. This is the first study to demonstrate invasion of human-RPEs, begin to characterize intracellular localization and survival of Pg within these cells. Collectively, invasion of RPE by Pg and its prolonged survival by autophagy evasion within these cells suggest a strong rationale for studying the link between oral infection and AMD pathogenesis in individuals with periodontitis.
Assuntos
Autofagossomos , Autofagia , Infecções por Bacteroidaceae , Citosol , Porphyromonas gingivalis , Epitélio Pigmentado da Retina , Vacúolos , Autofagossomos/metabolismo , Autofagossomos/microbiologia , Autofagossomos/ultraestrutura , Infecções por Bacteroidaceae/metabolismo , Infecções por Bacteroidaceae/microbiologia , Infecções por Bacteroidaceae/patologia , Linhagem Celular , Citosol/metabolismo , Citosol/microbiologia , Citosol/ultraestrutura , Humanos , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/ultraestrutura , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/microbiologia , Epitélio Pigmentado da Retina/ultraestrutura , Vacúolos/microbiologia , Vacúolos/patologia , Vacúolos/ultraestruturaRESUMO
PURPOSE: Fibrotic choroidal neovascular membranes (CNV) are the end-stage outcomes of neovascular age-related macular degeneration (AMD). No treatment is currently available for fibrotic CNV. We investigated the role of focal thermal laser ablation of the perfusing afferent arteriole as determined by dynamic indocyanine green angiography (ICGA). METHODS: We conducted a retrospective study of 20 patients with fibrotic CNV associated with significant subretinal fluid or retinal edema, who also demonstrated well-defined perfusing arterioles by dynamic ICGA. Patients underwent focal thermal laser occlusion of the perfusing afferent arteriole. Six, 12 and 24 weeks post-treatment, eyes underwent repeat examination with optical coherence tomography (OCT) and visual acuity testing, and ICGA at 12 weeks. RESULTS: Therapeutic closure of the perfusing afferent arterioles was achieved in 17 of 20 eyes immediately post-treatment. All 17 of these eyes demonstrated significant resolution of retinal edema and subretinal fluid, as evidenced by OCT, which was dramatic in some cases. Seven eyes demonstrated an improvement in visual acuity of 1 line or more. While most eyes demonstrated reperfusion within 3 months, many lesions suggested reduced vascularity and flow. CONCLUSION: Eyes with fibrotic CNV and associated retinal edema often demonstrate well-defined vascularity of the fibrosis with discrete perfusing arterioles when imaged by dynamic ICGA. Thermal laser occlusion of these arterioles can result in resolution of subretinal fluid, and occasionally an improvement in vision. This represents a potential therapeutic intervention for an advanced stage of AMD currently regarded as stable.
Assuntos
Neovascularização de Coroide/diagnóstico , Neovascularização de Coroide/cirurgia , Corantes , Verde de Indocianina , Fotocoagulação a Laser , Idoso , Arteríolas/cirurgia , Corioide/irrigação sanguínea , Neovascularização de Coroide/complicações , Neovascularização de Coroide/fisiopatologia , Feminino , Fibrose , Humanos , Masculino , Papiledema/complicações , Estudos Retrospectivos , Tomografia de Coerência Óptica , Acuidade VisualRESUMO
PURPOSE: To determine the impact of repetitive nonlethal oxidant injury with hydroquinone (HQ) on regulation of cell membrane blebbing and molecules, which are essential in extracellular matrix turnover (ECM) maintenance, especially matrix metalloproteinase (MMP)-2, tissue inhibitor of MMP (TIMP)-2, and type IV collagen in cultured RPE. In addition, to determine whether chronic oral HQ causes induction of sub-RPE deposit formation in a mouse model. METHODS: An ARPE-19 cell line stably expressing membrane-targeted green fluorescent protein (GFP) was challenged by exposure to HQ (100 microM). Repetitive acute (6 hours every 3 days for 4 weeks) or transient (6 hours followed by a recovery phase, every 5 days for 6 weeks) exposure to HQ were evaluated. An MTS assay, cell counts, and bromodeoxyuridine (BrdU) incorporation were used to detect cell viability and proliferation. Supernatants and cell homogenates were collected to assess MMP-2 and TIMP-2 activity by zymography and reverse zymography, proteins by Western blot, and type IV collagen accumulation by ELISA and immunostaining. Expression of MMP-2 and type IV collagen was examined by real-time RT-PCR on total RNA. Sixteen-month-old C57BL/6 female mice were fed a regular fat diet, with or without HQ (0.8%) in the drinking water, for 4 months. The eyes were removed for transmission electron microscopy of the retina and choroid after treatment. Semiquantitative grading of deposit severity was performed. RESULTS: In vitro, high doses of HQ (400-250 microM) killed a significant fraction of RPE cells ( approximately 60% of control). Low doses (50-100 microM) were nonlethal but induced significant blebbing. Both nonlethal repetitive acute and transient exposure to HQ were associated with diminished MMP-2 activity and increased collagen type IV accumulation. In vivo, mice exposed to oral HQ demonstrated moderately thick basal laminar deposits and a variable degree of deposits within Bruch's membrane (BrM). These homogeneous sub-RPE deposits accumulated in the eyes, consistent with early laminar deposits. CONCLUSIONS: In cultured RPE, nonlethal injury with HQ upregulated nonlethal blebbing and decreased ECM turnover. Similarly, in vivo exposure to oral HQ induced nonlethal bleb injury and sub-RPE deposits. These data support the hypothesis that HQ may regulate blebbing and molecules that influence ECM turnover. This study suggests that HQ may be another type of oxidant that causes injury to the RPE and may explain the association between environmental oxidants and early AMD.
Assuntos
Colágeno Tipo IV/metabolismo , Matriz Extracelular/efeitos dos fármacos , Hidroquinonas/toxicidade , Metaloproteinase 2 da Matriz/metabolismo , Mutagênicos/toxicidade , Epitélio Pigmentado Ocular/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Animais , Western Blotting , Bromodesoxiuridina/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Corioide/efeitos dos fármacos , Corioide/ultraestrutura , Colágeno Tipo IV/genética , Dieta , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/ultraestrutura , RNA Mensageiro/metabolismo , Retina/efeitos dos fármacos , Retina/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: Oxidative injury to the retinal pigment epithelium (RPE) has been proposed to be an important injury stimulus relevant to the accumulation of subretinal deposits in age-related macular degeneration (AMD). Cigarette smoking is a major risk factor for AMD, and cigarette smoke-related tar contains high concentrations of a potent oxidant, hydroquinone (HQ). This study was an investigation of the effects of cigarette smoke (CS) and HQ in the development of sub-RPE deposits in an experimental mouse model. METHODS: Sixteen-month-old C57BL/6 female mice were fed a high-fat diet (HFD) for 4.5 months. Mice were divided into two major experimental groups, one to examine the effects of cigarette smoke and one to study the effects of a defined cigarette smoke component such as HQ. In the first group, mice eyes were exposed to blue-green light (positive controls) or to whole cigarette smoke. A third group with no intervention served as the negative control. In the second experimental group, animals received a purified diet with HQ (0.8%) with low or high fat content for 4.5 months. Mice in both groups were euthanatized at 4.5 months and eyes processed for transmission electron microscopy. RESULTS: As previously demonstrated by our laboratory and others, most mice fed an HFD without other oxidant exposure demonstrated normal morphology or, in a few cases, small nodular basal laminar deposits. Eyes of mice exposed to whole cigarette smoke or to HQ in the food demonstrated a variable degree of basal laminar deposits and diffusely thickened Bruch's membrane. The choriocapillaris endothelium was variably hypertrophic. CONCLUSIONS: Exposure to cigarette smoke or the smoke-related redox molecule, HQ, results in the formation of sub-RPE deposits, thickening of Bruch's membrane, and accumulation of deposits within Bruch's membrane. Smoke-related oxidants may be another oxidative injury stimulus to the choriocapillaris and RPE, and may explain the association between cigarette smoking and early AMD.
Assuntos
Lâmina Basilar da Corioide/efeitos dos fármacos , Modelos Animais de Doenças , Degeneração Macular/etiologia , Oxidantes/efeitos adversos , Epitélio Pigmentado Ocular/efeitos dos fármacos , Fumar/efeitos adversos , Animais , Membrana Basal/ultraestrutura , Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/ultraestrutura , Cotinina/sangue , Gorduras na Dieta/administração & dosagem , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Feminino , Hidroquinonas/sangue , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/ultraestrutura , Retina/efeitos da radiaçãoRESUMO
Severe visual loss in patients with age-related macular degeneration is associated with the development of choroidal neovascularization (CNV). The pathogenic mechanisms for CNV formation have been extensively investigated, but remarkably little research has addressed the mechanisms for dysfunction of the retina in CNV. Using laser-induced CNV in mice, we evaluated the mechanisms of retinal dysfunction. At 3 days, 1 week, 2 weeks, and 4 weeks after laser application, retinas under experimental CNV were characterized physiologically (ERG recordings, synaptic uptake of the exocytotic marker FM1-43, and light-induced translocation of transducin), histologically, and immunohistochemically. ERG amplitudes were reduced by 20% at 1 week after CNV. Depolarization-induced FM1-43 uptake in photoreceptor synapses was selectively reduced by 45% at 1 week after CNV. Although photoreceptor outer segments were shortened by 36%, light adaptation as measured by transducin translocation was mostly preserved. Early in CNV (3 days to 1 week), Muller cells demonstrated induction of c-fos and pERK expression. Also, the density of macrophage-like, F4/80 immunoreactive cells increased approximately 3-fold. Minimal photoreceptor death occurred during the first week, and was variable thereafter. At later times in CNV formation (> or =2 weeks), expression of photoreceptor synaptic markers was reduced in the outer plexiform layer, indicating loss of photoreceptor synaptic terminals. ERG amplitudes, synaptic uptake of FM1-43, and the induction of c-fos and pERK in Muller cells were altered within 1 week of experimental CNV, suggesting that during CNV formation, deficits in retinal function, in particular photoreceptor synaptic function, precede degeneration of photoreceptor terminals and photoreceptor cell death.
Assuntos
Neovascularização de Coroide/fisiopatologia , Degeneração Neural/fisiopatologia , Células Fotorreceptoras/fisiopatologia , Sinapses/fisiologia , Adaptação Ocular/fisiologia , Sistema X-AG de Transporte de Aminoácidos/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Contagem de Células/métodos , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Eletrorretinografia/métodos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica/métodos , Lasers/efeitos adversos , Luz , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Compostos de Piridínio , Compostos de Amônio Quaternário , Receptores do Fator de Necrose Tumoral/metabolismo , Retina/metabolismo , Retina/patologia , Retina/fisiopatologia , Fatores de Tempo , Receptor fasRESUMO
PURPOSE: Dietary fat intake has been proposed as a mechanism of sub-RPE deposit formation. It has been demonstrated recently that sub-RPE deposits develop in 16- to 18-month-old C57BL/6 mice fed a high-fat diet and exposed to blue-green light. Hyperlipidemia also develops in these mice after they consume a high-fat diet. Because hyperlipidemia also develops in young C57BL/6 mice that overexpress APO B100, the major apolipoprotein in LDL cholesterol, this research was conducted to determine whether high-fat diet and plasma hyperlipidemia correlate with formation of basal laminar deposits (BLD) in young transgenic mice. METHODS: APO B100 and wild-type C57BL/6 2-month-old mice were fed a high-fat diet for 4.5 months. After the first month, the right eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light (20 mJ of argon 488 nm) over 2 weeks. Three months later, transmission electron microscopy (TEM) of the retina was performed to evaluate whether sub-RPE deposits correlate with plasma cholesterol and triglyceride levels. Several eyes were stained with filipin to detect cholesterol and osmium-thiocarbohydrazide-osmium (OTO) to detect neutral lipids in Bruch's membrane (BrM). A third group of APO B100 2-month-old mice were pretreated with vitamin E subcutaneously twice a week throughout the experiment and underwent the same light-exposure protocol. RESULTS: Mice fed a high-fat diet had a more elevated plasma triglyceride and cholesterol level than those that consumed a regular diet. Young APO B100 mice fed a high-fat diet had blood lipid levels higher than those in young wild-type mice that consumed high-fat diets, and these two groups had higher lipid levels than animals with regular diets, as shown previously in wild-type C57BL/6 (old and young). Eyes of APO B100 mice treated with blue-green light showed a high frequency of "moderate BLD", whereas the nonexposed eyes did not. In contrast, no BLD formed in either eye of the wild-type young mice fed a high-fat diet. In individual affected mice, only a weak correlation was observed between deposit severity and plasma lipid concentration. None of the eyes in mice with sustained hyperlipidemia with or without BLD demonstrated obvious widespread neutral lipid or cholesterol deposition in BLD or BrM. However, vitamin E-treated mice showed minimal formation of BLD. CONCLUSIONS: Although a high-fat diet is a necessary precondition for this model of BLD, the findings demonstrated a convincing direct correlation between plasma lipidemia and deposit severity. The results suggest that age, as shown in previous studies, and high-fat predispose to formation of BLD by altering hepatic and/or RPE lipid metabolism in ways more complicated than plasma hyperlipidemia alone.
Assuntos
Apolipoproteínas B/metabolismo , Lâmina Basilar da Corioide/metabolismo , Gorduras na Dieta/administração & dosagem , Degeneração Macular/metabolismo , Retina/efeitos da radiação , Vitamina E/uso terapêutico , Animais , Apolipoproteína B-100 , Apolipoproteínas B/genética , Lâmina Basilar da Corioide/ultraestrutura , Colesterol/sangue , Modelos Animais de Doenças , Filipina/metabolismo , Hiperlipidemias/sangue , Hiperlipidemias/etiologia , Luz , Degeneração Macular/patologia , Degeneração Macular/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Epitélio Pigmentado Ocular/metabolismo , Epitélio Pigmentado Ocular/ultraestrutura , Triglicerídeos/sangueRESUMO
PURPOSE: The pathogenesis of choroidal neovascularization (CNV) is postulated to be driven by angiogenesis, a process in which the cellular components of the new vessel complex are derived from cells resident within an adjacent preexisting capillary. Recently, an alternative paradigm, termed postnatal vasculogenesis, has been shown to contribute to some forms of neovascularization. In vasculogenesis, the cellular components of the new vessel complex are derived from circulating vascular progenitors from bone marrow. In the current study, transplantation of green fluorescent protein (GFP)-labeled bone marrow and laser-induced CNV were combined to examine the contribution of vasculogenesis to the formation of CNV. METHODS: Ten adult C57BL/6 female mice were used as recipients for bone marrow transplantation. Bone marrow was obtained from three C57BL/6 female mice transgenic for the beta-actin promoter GFP. One month after bone marrow transplantation, CNV was induced in recipient mice by making four separate burns in the choroid of each eye with a red diode laser. Four weeks after CNV was induced, eyes of recipient mice were processed for immunohistochemistry to detect GFP and markers for vascular smooth muscle cells (alpha-smooth muscle actin, desmin, and NG2 chondroitin sulfate proteoglycan), endothelial cells (CD31, BS-1 lectin), or macrophages (F4/80). RESULTS: GFP-labeled cells represented 17% of the total cell population in the lesion. Many of the GFP-labeled cells were immunoreactive for alpha-smooth muscle actin (39%), desmin, NG2, CD31 (41%), BS-1 lectin, or F4/80. GFP-labeled cells were morphologically indistinguishable from cells normally present in CNV lesions. CONCLUSIONS: This study is the first to demonstrate that bone marrow-derived progenitor cells are a source of endothelial and smooth musclelike cells in CNV.
Assuntos
Neovascularização de Coroide/etiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Actinas/metabolismo , Animais , Antígenos/metabolismo , Antígenos de Diferenciação/metabolismo , Biomarcadores , Corioide/irrigação sanguínea , Corioide/cirurgia , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Desmina/metabolismo , Endotélio Vascular/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde , Indicadores e Reagentes/metabolismo , Fotocoagulação a Laser , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Proteoglicanas/metabolismoRESUMO
PURPOSE: Cigarette smoking is the strongest environmental risk factor for all forms of age-related macular degeneration (AMD). In the present study, the influence of nicotine on the severity of choroidal neovascularization (CNV) in a mouse model of neovascular AMD and its effects on vascular smooth muscle cells derived from mouse choroid were investigated. METHODS: A mouse model for CNV was used to study the effects of nicotine in young and middle-aged mice. Nicotine was administered orally in the drinking water to achieve serum levels consistent with those of chronic smokers. Hexamethonium, a nonspecific nicotinic receptor antagonist, was injected subconjunctivally to counteract the effects of nicotine. A mouse choroidal vascular smooth muscle cell line was exposed to nicotine, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), or a combination of one of the factors and nicotine. Cell growth was determined by cell counts, and the activity of matrix metalloproteinase (MMP)-2 and -9 was quantified by gel zymography. RESULTS: Nicotine administration resulted in increased size and vascularity of CNV, and older mice developed a greater relative increase than younger mice. This effect was blocked by subconjunctival hexamethonium. Choroidal vascular smooth muscle cells demonstrated a statistically significant increase in growth after exposure to a combination of PDGF and nicotine. Nicotine also reversed VEGF-induced suppression of MMP-2 activity. CONCLUSIONS: Nicotine increases size and severity of experimental CNV in the present mouse model, possibly by potentiating PDGF-mediated upregulation of proliferation of choroidal smooth muscle cells or by other mechanisms. These results suggest that non-neuronal nicotinic receptor activation probably mediates some of the harmful effects of cigarette smoking in wet AMD.
Assuntos
Corioide/efeitos dos fármacos , Neovascularização de Coroide/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Administração Oral , Animais , Divisão Celular/efeitos dos fármacos , Corioide/irrigação sanguínea , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Combinação de Medicamentos , Feminino , Angiofluoresceinografia , Hexametônio/farmacologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Antagonistas Nicotínicos/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
PURPOSE: Macrophage recruitment to the choroid has been proposed to contribute to the pathogenesis of choroidal neovascularization (CNV) in AMD. The study was conducted to determine whether treatment with clodronate liposomes (CL(2)MDP-lip), which cause depletion of blood monocytes and lymph node macrophages, diminishes the severity of neovascularization in a mouse model of laser-induced CNV. METHODS: Laser-induced CNV was performed in female 16-month-old C57BL/6 mice. Macrophages were depleted by use of CL(2)MDP-lip intraperitoneally and subcutaneously 72 and 24 hours before and every 2 to 3 days after laser injury. Control mice received injections of either PBS alone or PBS liposomes. Blood monocyte and choroidal macrophage depletion were documented by flow cytometry and choroidal flatmount preparation analysis, respectively. Two weeks after laser injury, mice were injected intravenously with fluoresceinated dextran. The right eyes were removed and prepared for flatmount analysis of CNV surface area (in relative disc areas or DA), vascularity (relative fluorescence), and cellularity (propidium iodide stain). The mice were then perfused with 10% formaldehyde, and the left eyes were removed for histopathology. The means of the various parameters for four CNV lesions per eye were calculated. Fluorescein angiography was also performed. RESULTS: Flow cytometry of circulating monocytes and immunohistochemical analysis of choroidal macrophage density confirmed the effective depletion of blood monocytes and choroidal macrophages respectively in CL(2)MDP-lip-treated mice. Compared with the control, flatmount analysis of macrophage depleted mice demonstrated a significant reduction in size of the CNV area (2.8 +/- 0.5 DA vs. 1.4 +/- 0.1 DA; P < 0.043). The treated group also revealed less vascularity (1.6 +/- 0.1 units vs. 1.1 +/- 0.0 units; P < 0.0092) and cellularity of CNV lesions (3.3 +/- 0.6 DA vs. 1.7 +/- 0.1 DA, P < 0.04). Histopathology revealed that, in the macrophage-depleted group, CNV was smaller in diameter (1270 +/- 73 pixels vs. 770 +/- 82 pixels, P < 0.0006) and thickness (120 +/- 7 pixels vs. 96 +/- 7 pixels, P < 0.019). CONCLUSIONS: Macrophage depletion using CL(2)MDP-lip reduces size, cellularity, and vascularity of CNV. This observation supports the hypothesis that macrophages contribute to the severity of CNV lesions.
Assuntos
Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Macrófagos/fisiologia , Animais , Antimetabólitos/administração & dosagem , Contagem de Células , Corioide/irrigação sanguínea , Neovascularização de Coroide/prevenção & controle , Ácido Clodrônico/administração & dosagem , Dextranos , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Fluoresceínas , Lipossomos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
PURPOSE: Estrogen status influences the incidence and severity of many diseases in women. Because women with early menopause appear at risk for worse ARMD, estrogen deficiency may also contribute to the onset or severity of ARMD in women. It has been observed that aged male C57BL/6 mice fed a high-fat diet and briefly exposed to blue-green light exhibit development of significant sub-RPE deposits and mild Bruch's membrane (BrM) thickening. This model was used in an attempt to delineate the role of gender and estrogen status in this model. METHODS: C57BL/6 male and female mice of 9 or 16 months were fed a high-fat diet for 4.5 months. Several groups of 9-month-old female mice underwent estrogen depletion by ovariectomy, with or without supplementation with exogenous 17beta-estradiol. After 4 weeks of a high-fat diet, the eyes were exposed to seven 5-second doses of nonphototoxic levels of blue-green light over 2 weeks. Three and a half months after cessation of blue light treatment, transmission electron microscopy was performed to assess severity of deposits, BrM changes, and choriocapillaris endothelial morphology. In some mice, gelatin zymography and Western blot analyses were performed on protein extracts of freshly isolated RPE to determine the effect of estrogen on matrix metalloproteinase (MMP)-2 activity in the RPE. RESULTS: Both male and female 16-month-old mice showed qualitatively similar basal laminar deposit morphology, but the severity of thickness, continuity, and content was significantly greater in female mice. Aged female mice also demonstrated a trend toward more severe endothelial changes and increased BrM thickening compared with age-matched male mice. Ovariectomized middle-aged mice showed more severe deposits than sham-surgery control animals. However, ovariectomized mice that received high-dose estrogen supplementation also showed significant deposits, although they had thinner BrMs than did the estrogen-deficient mice. Loss of RPE MMP-2 activity correlated with deposit severity, with estrogen-deficient mice expressing less MMP-2 than ovary-intact control mice. CONCLUSIONS: Female gender in aged mice and estrogen deficiency in middle-aged mice appears to increase the severity of sub-RPE deposit formation. Estrogen deficiency may increase susceptibility to formation of sub-RPE deposits by dysregulating turnover of BrM, contributing to collagenous thickening and endothelial changes. Estrogen supplementation at the dosages used in this study does not appear to protect against formation of sub-RPE deposits.
Assuntos
Estrogênios/deficiência , Doenças Retinianas/etiologia , Fatores Etários , Animais , Western Blotting , Lâmina Basilar da Corioide/ultraestrutura , Capilares/patologia , Corioide/irrigação sanguínea , Gorduras na Dieta/administração & dosagem , Endotélio Vascular/ultraestrutura , Estradiol/sangue , Matriz Extracelular/ultraestrutura , Feminino , Luz , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ovariectomia , Ovário/fisiologia , Epitélio Pigmentado Ocular/enzimologia , Epitélio Pigmentado Ocular/ultraestrutura , Doenças Retinianas/patologia , Fatores SexuaisRESUMO
PURPOSE: For many vascular diseases, aging appears to be an independent risk factor for severity of vascular complications, and blood vessels of aged individuals often demonstrate exaggerated repair responses to injury. This study was undertaken to determine the influence of aging on the severity of neovascularization in a mouse model of laser-induced choroidal neovascularization (CNV). METHODS: CNV was induced in young (2-month-old) and aged (16-month-old) C57BL/6 mice by making four separate choroidal burns in each eye with a diode red laser (650 nm). At 1, 2, and 4 weeks, the left eyes were removed for histopathology, and the right eyes were removed for flatmount analysis of CNV surface area, vascularity, and cellularity. RESULTS: Aged mice demonstrated a much larger area of CNV than did young mice (3.81 +/- 1.28 vs. 1.36 +/- 0.99 disc areas, P < 0.001) at 2 weeks, when the lesions showed maximum growth. Aged mice also demonstrated higher ratios for vascularity and cellularity of the CNV (1.34 +/- 0.06 vs. 1.03 +/- 0.11, P < 0.0001 and 4.06 +/- 1.19 vs. 1.91 +/- 0.81, P < 0.002 at 2 weeks, respectively). Histopathology revealed that CNV in older eyes was larger, thicker, and more cellular than in young eyes. CONCLUSIONS: In mice, age is associated with more severe CNV, defined as larger surface area, greater vascularity, and greater cellularity. Age-related systemic susceptibility factors, independent of local changes in the retina, may contribute to the greater severity of CNV in older than in younger individuals.
Assuntos
Envelhecimento/fisiologia , Neovascularização de Coroide/fisiopatologia , Animais , Contagem de Células , Corioide/irrigação sanguínea , Corioide/cirurgia , Neovascularização de Coroide/patologia , Feminino , Angiofluoresceinografia , Terapia a Laser , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Fatores de RiscoRESUMO
OBJECTIVE: To evaluate the activation state of macrophage function in patients with age-related macular degeneration (AMD) by quantifying the production of the proinflammatory and angiogenic factor tumor necrosis factor alpha (TNF-alpha) and by correlating its expression with dry and wet AMD. METHODS: Circulating monocytes were obtained from the blood of patients with AMD or age-matched control subjects by gradient centrifugation. The monocytes were then analyzed for either TNF-alpha release from cultured macrophages in response to retinal pigment epithelium-derived blebs and cytokines or TNF-alpha messenger RNA content by reverse transcriptase-polymerase chain reaction. RESULTS: In human monocytes obtained from controls and AMD patients, TNF-alpha was expressed by freshly isolated monocytes and produced by macrophages in culture after stimulation with retinal pigment epithelium-derived blebs. However, wide variability in TNF-alpha expression was observed among different patients. Patients with monocytes that expressed the greatest amount of TNF-alpha demonstrated higher prevalence of choroidal neovascularization. CONCLUSIONS: Both controls and AMD patients vary in the activation state (defined as TNF-alpha expression) of circulating monocytes. Partially active monocytes, defined as high TNF-alpha expression, may be a biomarker to identify patients at risk for formation of choroidal neovascularization. CLINICAL RELEVANCE: Early diagnostic testing may prove useful to detect those patients who will progress to the more severe complications of the disease.
Assuntos
Neovascularização de Coroide/metabolismo , Degeneração Macular/metabolismo , Monócitos/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Células Cultivadas , Neovascularização de Coroide/etiologia , Feminino , Humanos , Ativação de Macrófagos/fisiologia , Macrófagos/fisiologia , Degeneração Macular/complicações , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Fator de Necrose Tumoral alfa/genéticaRESUMO
PURPOSE: To determine if prior exposure to pathogens associated with vascular disease, cytomegalovirus, Chlamydia pneumoniae, and Helicobacter pylori correlates with neovascular age-related macular degeneration (AMD). DESIGN: An experimental study. SETTING: Institutional. Bascom Palmer Eye Institute, October 2001 to December 2002. PATIENT POPULATION: 150 patients (47 neovascular amd, 36 dry amd, and 67 non-amd controls) were included in the study. exclusion criteria included hiv infection, malignancy, recent acute illness requiring hospitalization within 6 months, or immunosuppressive illness. PROCEDURE: Serum samples were obtained for analysis of cytomegalovirus, chlamydia pneumoniae, and helicobacter pylori igg antibody titers by elisa. MAIN OUTCOME MEASURE: Comparison of the distribution of igg titers between patients with wet amd, dry amd, and controls. RESULTS: The average cytomegalovirus IgG titer was higher in patients with wet AMD versus controls (p = 0.02, Student t-test, two-tailed) and patients with dry AMD (p = 0.06). Twenty-six (55%) of 47 subjects with wet AMD had high cytomegalovirus IgG titers compared with 14 (39%) of 36 patients with dry AMD (odds ratio [OR] = 2.23, 95% confidence interval [CI] = 0.77 to 6.44) and 23 (34%) of 67 control patients (OR = 2.49, 95% CI = 0.98 to 6.33). There was no major difference in the distribution of titers for Chlamydia pneumoniae IgG and Helicobacter pylori IgG in wet and dry AMD patients. Five of 47 patients with wet AMD (11%) had high antibody titers to all three pathogens, compared with only 1 of 36 patients with dry AMD (3%) (OR = 4.17, 95% CI = 0.46 to 37.36). CONCLUSIONS: There was a significant association of high cytomegalovirus IgG titer with neovascular AMD compared with dry AMD and control patients. Chronic infection with cytomegalovirus may be a novel risk factor for the progression from dry to neovascular AMD.
Assuntos
Anticorpos Antivirais/sangue , Neovascularização de Coroide/virologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/imunologia , Degeneração Macular/virologia , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antibacterianos/sangue , Chlamydophila pneumoniae/imunologia , Neovascularização de Coroide/imunologia , Infecções por Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Helicobacter pylori/imunologia , Humanos , Imunoglobulina G/análise , Degeneração Macular/imunologia , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
PURPOSE: Neovascular remodeling (NVR), the progression of small capillaries into large-caliber arterioles with perivascular fibrosis, represents a major therapeutic challenge in neovascular age-related macular degeneration (AMD). Neovascular remodeling occurs after laser-induced choroidal neovascularization (CNV) in aged but not young mice. Additionally, bone marrow-derived cells, including macrophages, endothelial precursor cells, and mesenchymal precursor cells, contribute to CNV severity. In this study, we investigated the impact of aged bone marrow transplantation (BMT) on the degree of fibrosis, size, and vascular morphology of CNV lesions in a mouse model of laser-induced CNV. METHODS: Young (2 months) and old (16 months) mice were transplanted with green fluorescent protein (GFP)-labeled bone marrow isolated from either young or old donors. Laser CNV was induced 1 month following transplant, and eyes were analyzed via choroidal flat mounts and immunohistochemistry 1 month postlaser. The identity of cells infiltrating CNV lesions was determined using specific markers for the labeled transplanted cells (GFP+), macrophages (F4/80+), perivascular mesenchymal-derived cells (smooth muscle actin, SMA+), and endothelial cells (CD31+). RESULTS: Bone marrow transplantation from aged mice transferred susceptibility to NVR into young recipients. Inversely, transplantation of young marrow into old mice prevented NVR, preserving small size and minimal fibrosis. Mice with NVR demonstrated a greater relative contribution of marrow-derived SMA+ perivascular mesenchymal cells as compared to other cells. CONCLUSIONS: Our findings indicate that the status of bone marrow is an important determining factor of neovascular severity. Furthermore, we find that perivascular mesenchymal cells, rather than endothelial cells, derived from aged bone marrow may contribute to increased CNV severity in this murine model of experimental neovascularization.