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1.
Exp Eye Res ; 184: 234-242, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31075224

RESUMO

The human retina is a complex tissue responsible for detecting photons of light and converting information from these photons into the neurochemical signals interpreted as vision. Such visual signaling not only requires sophisticated interactions between multiple classes of neurons, but also spatially-dependent molecular specialization of individual cell types. In this study, we performed single-cell RNA sequencing on neural retina isolated from both the fovea and peripheral retina in three human donors. We recovered a total of 8,217 cells, with 3,578 cells originating from the fovea and 4,639 cells originating from the periphery. Expression profiles for all major retinal cell types were compiled, and differential expression analysis was performed between cells of foveal versus peripheral origin. Globally, mRNA for the serum iron binding protein transferrin (TF), which has been associated with age-related macular degeneration pathogenesis, was enriched in peripheral samples. Cone photoreceptor cells were of particular interest and formed two predominant clusters based on gene expression. One cone cluster had 96% of cells originating from foveal samples, while the second cone cluster consisted exclusively of peripherally isolated cells. A total of 148 genes were differentially expressed between cones from the fovea versus periphery. Interestingly, peripheral cones were enriched for the gene encoding Beta-Carotene Oxygenase 2 (BCO2). A relative deficiency of this enzyme may account for the accumulation of carotenoids responsible for yellow pigment deposition within the macula. Overall, this data set provides rich expression profiles of the major human retinal cell types and highlights transcriptomic features that distinguish foveal and peripheral cells.


Assuntos
Fóvea Central/citologia , Perfilação da Expressão Gênica , Retina/citologia , Células Fotorreceptoras Retinianas Cones/citologia , Análise de Sequência de RNA , Idoso de 80 Anos ou mais , Dioxigenases/genética , Feminino , Fóvea Central/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Doadores de Tecidos , Transferrina/genética
2.
Eye (Lond) ; 31(1): 10-25, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27716746

RESUMO

Age-related macular degeneration (AMD) is a devastating disease-causing vision loss in millions of people around the world. In advanced stages of disease, death of photoreceptor cells, retinal pigment epithelial cells, and choroidal endothelial cells (CECs) are common. Loss of endothelial cells of the choriocapillaris is one of the earliest detectable events in AMD, and, because the outer retina relies on the choriocapillaris for metabolic support, this loss may be the trigger for progression to more advanced stages. Here we highlight evidence for loss of CECs, including changes to vascular density within the choriocapillaris, altered abundance of CEC markers, and changes to overall thickness of the choroid. Furthermore, we review the key components and functions of the choroid, as well as Bruch's membrane, both of which are vital for healthy vision. We discuss changes to the structure and molecular composition of these tissues, many of which develop with age and may contribute to AMD pathogenesis. For example, a crucial event that occurs in the aging choriocapillaris is accumulation of the membrane attack complex, which may result in complement-mediated CEC lysis, and may be a primary cause for AMD-associated choriocapillaris degeneration. The actions of elevated monomeric C-reactive protein in the choriocapillaris in at-risk individuals may also contribute to the inflammatory environment in the choroid and promote disease progression. Finally, we discuss the progress that has been made in the development of AMD therapies, with a focus on cell replacement.


Assuntos
Envelhecimento/fisiologia , Corioide/patologia , Degeneração Macular/patologia , Lâmina Basilar da Corioide/patologia , Capilares/patologia , Corioide/irrigação sanguínea , Células Endoteliais/patologia , Humanos , Epitélio Pigmentado da Retina/patologia , Fatores de Risco
3.
Gene Ther ; 21(7): 662-72, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24807808

RESUMO

Mutations in CEP290 are the most common cause of Leber congenital amaurosis (LCA), a severe inherited retinal degenerative disease for which there is currently no cure. Autosomal recessive CEP290-associated LCA is a good candidate for gene replacement therapy, and cells derived from affected individuals give researchers the ability to study human disease and therapeutic gene correction in vitro. Here we report the development of lentiviral vectors carrying full-length CEP290 for the purpose of correcting the CEP290 disease-specific phenotype in human cells. A lentiviral vector containing CMV-driven human full-length CEP290 was constructed. Following transduction of patient-specific, iPSC-derived, photoreceptor precursor cells, reverse transcriptase-PCR analysis and western blotting revealed vector-derived expression. As CEP290 is important in ciliogenesis, the ability of fibroblast cultures from CEP290-associated LCA patients to form cilia was investigated. In cultures derived from these patients, fewer cells formed cilia compared with unaffected controls. Cilia that were formed were shorter in patient-derived cells than in cells from unaffected individuals. Importantly, lentiviral delivery of CEP290 rescued the ciliogenesis defect. The successful construction and viral transfer of full-length CEP290 brings us closer to the goal of providing gene- and cell-based therapies for patients affected with this common form of LCA.


Assuntos
Antígenos de Neoplasias/genética , Células-Tronco Pluripotentes Induzidas/transplante , Amaurose Congênita de Leber/terapia , Lentivirus/genética , Proteínas de Neoplasias/genética , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Cílios/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Vetores Genéticos/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/patologia , Camundongos , Proteínas de Neoplasias/metabolismo , Retina/patologia , Transdução Genética
4.
Exp Eye Res ; 111: 61-6, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23567206

RESUMO

Lipofuscin accumulation has been observed in a number of neurodegenerative diseases. We recently found that autofluorescent particles also occur in the aged human optic nerve. In this study we sought to determine the nature of these particles and their correlation with aging, age-related macular degeneration (AMD) and primary open angle glaucoma (POAG). Groups of eight optic nerves from patients diagnosed with primary open angle glaucoma, age-related macular degeneration, age-matched controls and four optic nerves derived from controls younger than 42 years were used for the study. All samples were fixed in paraformaldehyde and frozen frontal sections were prepared. Sections were analyzed with fluorescence microscopy, bright field microscopy, Sudan black staining and spectrofluorometry using a confocal laser scanning microscope. Sections were photographed and analyzed to establish the distribution, quantity, and size of the autofluorescent particles. Additionally, transmission electron microscopy was used to determine the ultrastructural location of the granules. On unstained sections under light microscopy granules are detectable as pale brown inclusions and are easily stained with oil-soluble dyes, such as Sudan black. Granules fluoresce when excited at all tested wavelengths but lose their fluorescence after staining with Sudan black. These particles are distributed throughout the axonal columns, but not in the septa, and appear to be located within the glia ensheathing optic nerve axons. The histologic properties of the granules seen in the optic nerve sections correspond to lipofuscin aggregates, a result of incomplete degradation of oxidized proteins. Our morphometric analyses indicate that overall the optic nerves from control, glaucoma, and AMD donors contain similar amounts of lipofuscin. However, optic nerves derived from donors with glaucoma contain lipofuscin particles that are larger than those observed in the age-matched control and AMD groups. Furthermore optic nerves from glaucoma donors display a smaller diameter than those from age-matched controls resulting in a higher concentration of lipofuscin in glaucomatous optic nerves.


Assuntos
Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/patologia , Lipofuscina/metabolismo , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Adulto , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Envelhecimento/patologia , Axônios/metabolismo , Axônios/patologia , Feminino , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Microscopia Eletrônica de Transmissão , Nervo Óptico/ultraestrutura
5.
Ann Burns Fire Disasters ; 24(2): 89-93, 2011 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-22262966

RESUMO

Introduction. The key element of a safe workplace for employees is the maintenance of fire safety. Thermal, chemical, and electrical burns are common types of burns at the workplace. This study assessed the epidemiology of work-related burn injuries on the basis of the workers treated in a regional burn centre. Methods. Two years' retrospective data (2005-2006) from the Trauma Registry of the American College of Surgeons of the Joseph M. Still Burn Center at Doctors Hospital in Augusta, Georgia, were collected and analysed. Results. During the time period studied, 2510 adult patients with acute burns were admitted; 384 cases (15%) were work-related. The average age of the patients was 37 yr (range, 15-72 yr). Males constituted the majority (90%) of workrelated burn injury admissions. The racial distribution was in accordance with the Centre's admission census. Industrial plant explosions accounted for the highest number of work-related burns and, relatively, a significant number of patients had chemical burns. The average length of hospital stay was 5.54 days. Only three patients did not have health insurance and four patients (1%) died. Conclusion. Burn injuries at the workplace predominantly occur among young male workers, and the study has shown that chemical burns are relatively frequent. This study functions as the basis for the evaluation of work-related burns and identification of the causes of these injuries to formulate adequate safety measures, especially for young, male employees working with chemicals.

6.
Hum Mutat ; 30(8): 1183-8, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19431183

RESUMO

To assist in distinguishing disease-causing mutations from nonpathogenic polymorphisms, we developed an objective algorithm to calculate an "estimate of pathogenic probability" (EPP) based on the prevalence of a specific variation, its segregation within families, and its predicted effects on protein structure. Eleven missense variations in the RPE65 gene were evaluated in patients with Leber congenital amaurosis (LCA) using the EPP algorithm. The accuracy of the EPP algorithm was evaluated using a cell-culture assay of RPE65-isomerase activity The variations were engineered into plasmids containing a human RPE65 cDNA and the retinoid isomerase activity of each variant was determined in cultured cells. The EPP algorithm predicted eight substitution mutations to be disease-causing variants. The isomerase catalytic activities of these RPE65 variants were all less than 6% of wild-type. In contrast, the EPP algorithm predicted the other three substitutions to be non-disease-causing, with isomerase activities of 68%, 127%, and 110% of wild-type, respectively. We observed complete concordance between the predicted pathogenicities of missense variations in the RPE65 gene and retinoid isomerase activities measured in a functional assay. These results suggest that the EPP algorithm may be useful to evaluate the pathogenicity of missense variations in other disease genes where functional assays are not available.


Assuntos
Proteínas de Transporte/genética , Proteínas do Olho/genética , Mutação de Sentido Incorreto , Algoritmos , Sequência de Aminoácidos , Sequência de Bases , Biocatálise , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Linhagem Celular , Primers do DNA , DNA Complementar , Proteínas do Olho/química , Proteínas do Olho/fisiologia , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Linhagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa , cis-trans-Isomerases
7.
Eye (Lond) ; 23(4): 747-55, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18600240

RESUMO

The events that lead to choroidal neovascularization in eyes with age-related macular degeneration are poorly understood. One possibility that has been explored in a number of studies is that macrophages can promote neovascular changes. In this paper, we summarize the evidence for inflammation in general and macrophages in particular in pathologic neovascularization, and discuss how the diverse functions of these cells may promote or inhibit macular disease. We also discuss some of the conflicting findings regarding the role of macrophages in experimental choroidal neovascularization in mouse models, and suggest areas for future research.


Assuntos
Neovascularização de Coroide/patologia , Macrófagos/fisiologia , Degeneração Macular/patologia , Animais , Corioide/imunologia , Corioide/patologia , Neovascularização de Coroide/imunologia , Neovascularização de Coroide/fisiopatologia , Modelos Animais de Doenças , Humanos , Imuno-Histoquímica , Inflamação/fisiopatologia , Macrófagos/patologia , Degeneração Macular/imunologia , Degeneração Macular/fisiopatologia , Camundongos
8.
Ann Burns Fire Disasters ; 22(4): 200-2, 2009 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-21991182

RESUMO

Anticipating functional outcomes of patients managed in an in-patient burn wound centre can help in advising patients and their families of prognosis as well as assist case managers in discharge planning. The records of 37 burn patients were reviewed; one patient expired and was removed from further analysis. Data were obtained regarding patient characteristics, types and locations of burns and other wounds, ventilator use, level of mobility at hospital discharge, and disposition; three patients lacked discharge ambulation status and were removed from the outcome comparison analysis. Of the 36 patients, 17 had thermal burns and nine (25%) had associated inhalation injuries. Thermal burn patients (p = 0.02), patients requiring ventilator support during their hospital stay (p = 0.04), and those with inhalation injuries (p = 0.04) were less likely to be ambulating independently or with assistance at discharge from the burn wound centre than other patients. This preliminary study suggests that patients with thermal burns and inhalation injuries and those requiring ventilator support were less likely to be ambulatory at hospital discharge. Further studies appear indicated.

9.
Ann Burns Fire Disasters ; 21(3): 153-5, 2008 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-21991129

RESUMO

The commonest cause of carpal tunnel syndrome (CTS) is a congenital predisposition - the carpal tunnel is simply narrower in some people than in others. The development of CTS due to various burn causes has never been reported. This study describes some demographic features of all reported CTS cases following different types of burn in patients admitted to our burns centre in the USA. A retrospective case study was carried out to identify CTS following different types of burns among the patients admitted between January 2001 and January 2006. A descriptive data analysis was carried out to observe CTS following hand burns. There were 36 CTS cases in 28 patients presenting various kinds of burns in the upper extremities. The mean age of the CTS patients was 52 years (SD, 10.51 yr) and 79% were male. Of these, 57% had sustained thermal burns, 32% electrical burns, and the remainder had scalds or chemical burns. More patients (56%) had second-degree burns than third-degree burns and all but one of the patients with electrical burns had second-degree burns. This observation demonstrates that there were a considerable number of CTS cases following thermal burn injury compared to previous reporting. This study also suggests the need of a prospective study to examine the association between burns in the upper extremities and the likelihood of their progression to CTS and whether any specific type of burn is more likely to result in CTS.

10.
Exp Eye Res ; 85(1): 34-43, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17477921

RESUMO

Best vitelliform macular dystrophy (BMD) is an autosomal dominant inherited macular degenerative disease caused by mutations in the gene BEST1 (formerly VMD2). Prior reports indicate that BMD is characterized histopathologically by accumulation of lipofuscin in the retinal pigment epithelium (RPE). However, this accumulation has not been quantified and the chemical composition of lipofuscin in BMD has not been examined. In this study we characterize the histopathology of a donor eye from a rare individual homozygous for a mutation (W93C) in BEST1. We find that this individual's disease was not any more severe than has been described for heterozygotes. We then used this tissue to quantify lipofuscin accumulation by enriching intracellular granules from RPE cells on sucrose gradients and counting the granules in each density fraction. Granules from the homozygous donor eye as well as a donor eye from an individual heterozygous for the mutation T6R were compared with age-matched control eyes. Interestingly, the least dense fraction, representing classical lipofuscin granules was either not present or significantly diminished in the BMD donor eyes and the autoflourescence associated with lipofuscin had shifted to denser fractions. However, a substantial enrichment for granules in fractions of higher density was also noted in the BMD samples. Inspection of granules from the homozygous donor eye by electron microscopy revealed a complex abnormal multilobular structure. Analysis of granules by HPLC indicated a approximately 1.6- and approximately fourfold overall increase in A2E in the BMD eyes versus age-matched control eyes, with a shift of A2E to more dense granules in the BMD donor eyes. Despite the increase in A2E and total intracellular granules, the RPE in the homozygous donor eyes was relatively well preserved. Based on these data we conclude that the clinical and histopathologic consequences to the homozygous donor were not any more severe than has been reported previously for individuals who are established or presumptive heterozygotes. We find that A2E is a component of the lipofuscin accumulated in BMD and that it is more abundant than in control eyes suggesting that the etiology of BMD is similar to Stargardt's disease and Stargardt-like macular dystrophy. Finally, the changes we observe in the granules suggest that the histopathology and eventual vision loss associated with BMD may be due to defects in the ability of the RPE to fully degrade phagocytosed photoreceptor outer segments.


Assuntos
Canais de Cloreto/genética , Proteínas do Olho/genética , Degeneração Macular/genética , Compostos de Piridínio/análise , Retinoides/análise , Idoso de 80 Anos ou mais , Bestrofinas , Olho/patologia , Angiofluoresceinografia/métodos , Heterozigoto , Homozigoto , Humanos , Imuno-Histoquímica/métodos , Lipofuscina/análise , Degeneração Macular/patologia , Masculino , Microscopia Eletrônica/métodos , Mutação/genética , Linhagem , Epitélio Pigmentado Ocular/química , Epitélio Pigmentado Ocular/patologia
11.
Prog Retin Eye Res ; 20(6): 705-32, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11587915

RESUMO

Age-related macular degeneration (AMD) is a blinding disease that afflicts millions of adults in the Western world. Although it has been proposed that a threshold event occurs during normal aging which leads to AMD, the sequelae of biochemical, cellular, and/or molecular events leading to the development of AMD are poorly understood. Although available data provide strong evidence that a significant proportion of AMD has a genetic basis, no gene(s) has yet been identified that causes a significant proportion of AMD. Moreover, no major molecular pathways involved in the etiology of this disease have been elucidated.Drusen, pathological deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane, are significant risk factors for the development of AMD. In our view, the development of testable new hypotheses of drusen origins has been hindered significantly by the absence of a comprehensive profile of their molecular composition. In this review, we describe an integrated ultrastructural, histochemical, molecular biological, and biochemical approach to identify specific molecular pathways associated with drusen biogenesis. The implicit assumption underlying these recent investigations has been that a thorough understanding of the composition of drusen and source(s) of drusen-associated material is likely to provide fresh insight into the pathobiology underlying AMD. Significantly, these studies have revealed that proteins associated with inflammation and immune-mediated processes are prevalent among drusen-associated constituents. Transcripts that encode a number of these molecules have been detected in retinal, RPE, and choroidal cells. These data have also lead to the observations that dendritic cells, potent antigen-presenting cells, are intimately associated with drusen development and that complement activation is a key pathway that is active both within drusen and along the RPE-choroid interface. We propose herein a unifying hypothesis of drusen biogenesis that attempts to incorporate a large body of new and previously published structural, histochemical, and molecular data pertaining to drusen composition and development. This theory is put forth with the acknowledgment that numerous AMD genotypes may exist. Thus, only some aspects of the proposed hypothesis may be involved in any given AMD genotype. Importantly, this hypothesis invokes, for the first time, the potential for a direct role of cell- and immune-mediated processes in drusen biogenesis. We acknowledge that the proposed hypothesis clearly represents a paradigm shift in our conceptualization pertaining to pathways that participate in the development of drusen and age-related macular degeneration. It is our hope that other investigators will test, validate and/or refute various aspects of this hypothesis, and in so doing, increase our overall understanding of the biological pathways associated with early AMD.


Assuntos
Envelhecimento/fisiologia , Lâmina Basilar da Corioide/imunologia , Degeneração Macular/imunologia , Epitélio Pigmentado Ocular/imunologia , Drusas Retinianas/imunologia , Biomarcadores , Lâmina Basilar da Corioide/patologia , Células Dendríticas/imunologia , Proteínas do Olho/metabolismo , Humanos , Sistema Imunitário , Degeneração Macular/etiologia , Degeneração Macular/patologia , Filosofia , Epitélio Pigmentado Ocular/patologia , Drusas Retinianas/complicações , Drusas Retinianas/patologia
12.
Hum Mol Genet ; 10(16): 1709-18, 2001 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-11487575

RESUMO

We have determined the molecular basis for Usher syndrome type 1F (USH1F) in two families segregating for this type of syndromic deafness. By fluorescence in situ hybridization, we placed the human homolog of the mouse protocadherin Pcdh15 in the linkage interval defined by the USH1F locus. We determined the genomic structure of this novel protocadherin, and found a single-base deletion in exon 10 in one USH1F family and a nonsense mutation in exon 2 in the second. Consistent with the phenotypes observed in these families, we demonstrated expression of PCDH15 in the retina and cochlea by RT-PCR and immunohistochemistry. This report shows that protocadherins are essential for maintenance of normal retinal and cochlear function.


Assuntos
Caderinas/genética , Surdez/genética , Mutação , Precursores de Proteínas/genética , Adulto , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Proteínas Relacionadas a Caderinas , Caderinas/análise , Cóclea/química , Análise Mutacional de DNA , Feminino , Feto , Perfilação da Expressão Gênica , Ligação Genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Dados de Sequência Molecular , Linhagem , Polimorfismo Conformacional de Fita Simples , Precursores de Proteínas/análise , Retina/química , Retina/embriologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Síndrome
13.
Eye (Lond) ; 15(Pt 3): 390-5, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11450763

RESUMO

PURPOSE: The ocular fundi of many patients with membranoproliferative glomerulonephritis type II (MPGN-II) are characterised by the presence of deposits within Bruch's membrane that resemble drusen, hallmark lesions associated with age-related macular degeneration (AMD). Glomerulonephritis (GN)-associated drusen appear at a younger age, however, than do drusen in individuals with AMD. In light of recent evidence that immune-mediated events participate in drusen biogenesis and AMD, we examined the structure and composition of drusen in eyes obtained from human donors with two distinct glomerulopathies, both of which involve complement deposition within glomeruli. These features were compared with those of drusen from patients with clinically documented AMD. METHODS: Eyes obtained from two human human donors diagnosed with membranous and post-streptococcal GN, respectively, were analysed histochemically, immunohistochemically and ultrastructurally. RESULTS: Subretinal pigment epithelial (RPE) deposits in both types of GN are numerous and indistinguishable, both structurally and compositionally, from drusen in donors with AMD. GN-associated drusen exhibit sudanophilia, bind filipin, and react with antibodies directed against vitronectin, complement C5 and C5b-9 complexes, TIMP-3 and amyloid P component. Drusen from the membranous GN donor, but not the post-streptococcal GN donor, reacted with peanut agglutinin and antibodies directed against MHC class II antigens and IgG. The ultrastructural characteristics of these deposits were also identical with those of AMD-associated drusen. CONCLUSIONS: The composition and structure of ocular drusen associated with membranous and post-streptococcal/segmental GN are generally similar to those of drusen in individuals with AMD. In view of the recent data supporting the involvement of complement activation in drusen biogenesis and the pathobiology of AMD, further studies of the biological relationships between AMD and diseases associated with complement activation are warranted.


Assuntos
Ativação do Complemento , Glomerulonefrite/complicações , Drusas Retinianas/patologia , Glomerulonefrite Membranosa/complicações , Humanos , Técnicas Imunoenzimáticas , Degeneração Macular/complicações , Microscopia Eletrônica , Pessoa de Meia-Idade , Epitélio Pigmentado Ocular/ultraestrutura , Drusas Retinianas/etiologia , Drusas Retinianas/imunologia , Infecções Estreptocócicas/complicações
14.
Am J Ophthalmol ; 131(6): 767-81, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11384575

RESUMO

PURPOSE: The inheritance of specific apolipoprotein E allelles has been linked to atherosclerosis, Alzheimer disease, and, most recently, to the incidence of age-related macular degeneration. Apolipoprotein E is a common component of the extracellular plaques and deposits characteristic of these disorders, including drusen, which are a hallmark of age-related macular degeneration. Accordingly, we assessed the potential biosynthetic contribution of local ocular cell types to the apolipoprotein E found in drusen. METHODS: We measured apolipoprotein E mRNA levels in human donor tissues using a quantitative assay of apolipoprotein E transcription, and we localized apolipoprotein E protein to specific cell types and compartments in the neural retina, retinal pigmented epithelium, and choroid using laser scanning confocal immunofluorescence microscopy. RESULTS: Apolipoprotein E immunoreactivity is associated with photoreceptor outer segments, the retinal ganglion cell layer, the retinal pigmented epithelium basal cytoplasm and basal lamina, and with both collagenous layers of Bruch membrane. Apolipoprotein E appears to be a ubiquitous component of drusen, irrespective of clinical phenotype. It also accumulates in the cytoplasm of a subpopulation of retinal pigmented epithelial cells, many of which overlie or flank drusen. Mean levels of apolipoprotein E mRNA in the adult human retina are 45% and 150% of the levels measured in liver and adult brain, the two most abundant biosynthetic sources of apolipoprotein E. Apolipoprotein E mRNA levels are highest in the inner retina, and lowest in the outer retina where photoreceptors predominate. Significant levels of apolipoprotein E mRNA are also present in the retinal pigmented epithelium/choroid complex and in cultured human retinal pigmented epithelial cells. CONCLUSIONS: Apolipoprotein E protein is strategically located at the same anatomic locus where drusen are situated, and the retinal pigmented epithelium is the most likely local biosynthetic source of apolipoprotein E at that location. Age-related alteration of lipoprotein biosynthesis and/or processing at the level of the retinal pigmented epithelium and/or Bruch membrane may be a significant contributing factor in drusen formation and age-related macular degeneration pathogenesis.


Assuntos
Apolipoproteínas E/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Retina/metabolismo , Adulto , Idoso , Apolipoproteínas E/genética , Lâmina Basilar da Corioide/metabolismo , Células Cultivadas , Corioide/metabolismo , Imunofluorescência , Humanos , Microscopia Confocal , Pessoa de Meia-Idade , Epitélio Pigmentado Ocular/citologia , RNA Mensageiro/metabolismo , Retina/citologia , Drusas Retinianas/etiologia , Drusas Retinianas/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Distribuição Tecidual
16.
FASEB J ; 14(7): 835-46, 2000 May.
Artigo em Inglês | MEDLINE | ID: mdl-10783137

RESUMO

Age-related macular degeneration (AMD), a blinding disorder that compromises central vision, is characterized by the accumulation of extracellular deposits, termed drusen, between the retinal pigmented epithelium and the choroid. Recent studies in this laboratory revealed that vitronectin is a major component of drusen. Because vitronectin is also a constituent of abnormal deposits associated with a variety of diseases, drusen from human donor eyes were examined for compositional similarities with other extracellular disease deposits. Thirty-four antibodies to 29 different proteins or protein complexes were tested for immunoreactivity with hard and soft drusen phenotypes. These analyses provide a partial profile of the molecular composition of drusen. Serum amyloid P component, apolipoprotein E, immunoglobulin light chains, Factor X, and complement proteins (C5 and C5b-9 complex) were identified in all drusen phenotypes. Transcripts encoding some of these molecules were also found to be synthesized by the retina, retinal pigmented epithelium, and/or choroid. The compositional similarity between drusen and other disease deposits may be significant in view of the recently established correlation between AMD and atherosclerosis. This study suggests that similar pathways may be involved in the etiologies of AMD and other age-related diseases.


Assuntos
Envelhecimento/metabolismo , Degeneração Macular/metabolismo , Proteínas/metabolismo , Drusas Retinianas/metabolismo , Amiloidose/metabolismo , Amiloidose/patologia , Arteriosclerose/metabolismo , Arteriosclerose/patologia , Sequência de Bases , Primers do DNA , Glomerulonefrite Membranoproliferativa/metabolismo , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Imuno-Histoquímica , Degeneração Macular/patologia , Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Drusas Retinianas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dermatopatias/metabolismo , Dermatopatias/patologia
17.
Am J Ophthalmol ; 129(2): 205-14, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10682974

RESUMO

PURPOSE: To determine whether basal laminar drusen differ in their location, ultrastructure, or composition from drusen associated with aging and age-related macular degeneration. METHODS: A paraffin-embedded block from an eye of a patient with basal laminar drusen was obtained. Sections were examined immunohistochemically using a battery of antibodies and lectins directed against drusen-associated proteins and glycoconjugates, respectively. Thin sections were examined by electron microscopy and compared with eyes with age-related macular degeneration. RESULTS: Drusen in the eye with basal laminar drusen are located between the basal lamina of the retinal pigment epithelium and the inner collagenous layer of Bruch membrane, just as they are in age-related macular degeneration. Two distinct ultrastructural phenotypes are observed in the eye with basal laminar drusen; their substructure is indistinguishable from drusen phenotypes in age-related macular degeneration. Both basal laminar drusen and drusen associated with age-related macular degeneration are bound by the lectins Ricinis communis agglutinin and Arachis hypogea agglutinin (after neuraminidase digestion) and by antivitronectin, anti-HLA-DR, anti-serum amyloid P, and anti-C5 antibodies, but not by antibodies directed against basement membrane-associated heparan sulfate proteoglycan, laminin, fibrinogen, or collagen type IV. CONCLUSIONS: These data support the notion that cuticular or basal laminar drusen are similar to, and perhaps indistinguishable from, drusen associated with age-related macular degeneration and are not nodular or diffuse thickenings of Bruch membrane, as previously suggested. Thus, we suggest basal laminar drusen is a misnomer. This clinical phenotype should be identified as "early adult onset, grouped drusen" or by the eponym "Gass syndrome." Features of basal laminar drusen, such as uniform drusen size, clustered distribution, and angiographic features, do not appear to be related to differences in drusen location, composition, or substructure.


Assuntos
Envelhecimento/patologia , Lâmina Basilar da Corioide/ultraestrutura , Degeneração Macular/patologia , Epitélio Pigmentado Ocular/ultraestrutura , Drusas Retinianas/patologia , Biomarcadores/análise , Lâmina Basilar da Corioide/metabolismo , Proteínas do Olho/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Glicoconjugados/metabolismo , Humanos , Técnicas Imunoenzimáticas , Degeneração Macular/complicações , Degeneração Macular/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Drusas Retinianas/complicações , Drusas Retinianas/metabolismo
18.
Invest Ophthalmol Vis Sci ; 40(13): 3305-15, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10586957

RESUMO

PURPOSE: To determine whether vitronectin (Vn), a plasma protein and extracellular matrix molecule that is also a prominent constituent of drusen, is synthesized by cells in the adult human retina. METHODS: The distribution of Vn in the normal adult human retina was examined using antibodies to circulating plasma Vn and to the multimeric, heparin-binding form that is most prevalent in extravascular tissues. Evidence of Vn transcription by retinal cells was analyzed by in situ hybridization and also by reverse transcription of total RNA derived from dissociated human or mouse photoreceptors followed by amplification using polymerase chain reaction (RT-PCR). RESULTS: Cytoplasmic immunoreactivity for plasma Vn or multimeric Vn was detected in photoreceptors, in a subpopulation of neurons situated in the inner retina, and in vitreous hyalocytes. Extracellular labeling was limited primarily to Bruch's membrane and the retinal vasculature. At the transcriptional level, Vn mRNA was localized to both photoreceptors and ganglion cells by in situ hybridization. The in situ findings were corroborated by RT-PCR using total RNA from dissociated mouse or human photoreceptor cells. CONCLUSIONS: The results constitute the first evidence for Vn gene expression by adult neurons in the mammalian central nervous system. The identification of the photoreceptors as a cellular source of Vn suggests that these cells have the potential to make a biosynthetic contribution to the Vn that is found in drusen.


Assuntos
Expressão Gênica , RNA Mensageiro/metabolismo , Retina/metabolismo , Vitronectina/genética , Adulto , Idoso , Animais , Primers do DNA/química , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Pessoa de Meia-Idade , Células Fotorreceptoras de Vertebrados/metabolismo , Células Ganglionares da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vitronectina/biossíntese
19.
Mol Vis ; 5: 28, 1999 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-10562652

RESUMO

Age-related macular degeneration (AMD) is characterized in part by the deposition of extracellular deposits, including drusen, in the aging macula. A number of clinical studies have revealed a strong association between the number, size, and degree of confluency of drusen and AMD. Although a number of distinct morphological classes, or phenotypes, of drusen can be resolved at the ultrastructural level, very little is known about the compositional and etiological relationship between these phenotypes. A number of recent studies have begun to provide insight into the composition of drusen at the light microscopic level of resolution. Out of 33 extracellular matrix proteins evaluated, vitronectin was identified in hard and soft drusen [FASEB J 1999; 13:477-84]. Drusen have also been found to contain carbohydrate moieties which are labeled by wheat germ agglutinin (WGA), and Limax flavus agglutinin (LFA). We have recently extended these histochemical, immunohistochemical, and biochemical investigations to examine the relationship between substructural drusen phenotype and composition. The initial results of these observations, generated from a repository of human donor eyes processed within four hours of death, are reported herein. Five distinct substructural drusen phenotypes were identified in tissue sections from eyes of approximately 400 donors; all five phenotypes were observed in eyes from donors with and without clinically documented AMD. Interestingly, no strict relationship between size (one important discriminator between "hard" and "soft" drusen class) and morphology was noted for four out of the five drusen phenotypes. Sections from the same donors were incubated with antibodies directed against vitronectin and with the lectins WGA and LFA, three probes recently shown to label hard and soft drusen at the light microscopic level of resolution. As anticipated, all of these probes bound to all phenotypes of drusen examined. These data suggest that different phenotypes of drusen, although they may differ significantly with respect to their substructural morphology, may possess a similar complement of extracellular matrix-associated proteins and saccharides. Ongoing investigations are directed toward determining whether there exist specific drusen constituents, not yet identified, that impart phenotypic and/or ontogenic specificity to drusen. It is anticipated that a more complete understanding of drusen composition, as it relates to phenotype, will provide significant new insight into the biology and etiology of various clinically manifested forms of AMD.


Assuntos
Drusas Retinianas/patologia , História do Século XIX , Humanos , Imuno-Histoquímica , Degeneração Macular/complicações , Degeneração Macular/patologia , Microscopia Eletrônica , Drusas Retinianas/classificação , Drusas Retinianas/complicações , Drusas Retinianas/história
20.
J Histochem Cytochem ; 47(12): 1533-40, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10567437

RESUMO

Ocular drusen are extracellular deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane. Although the presence of large and/or numerous drusen in the macula is a significant risk factor for development of age-related macular degeneration (AMD), a major cause of irreversible blindness, little is known about their origin or composition. We have expanded on our previous investigations related to drusen-associated glycoconjugates by examining lectin binding patterns after removal of terminal sialic acid residues. Strikingly, intense and distinct labeling of drusen subdomains is revealed by Arachea hypogea agglutinin (PNA) after neuraminidase treatment. PNA binding is confined to discrete domains within both hard and soft drusen. These "cores" are positioned centrally within drusen and are typically juxtaposed to Bruch's membrane. Only one core per druse is observed. PNA labeling of drusen cores does not co-localize with associated lipids and is abrogated by digestion with O-glycosidase but not N-glycosidase. The association of cores with small drusen suggests that they may participate in drusen biogenesis. (J Histochem Cytochem 47:1533-1539, 1999)


Assuntos
Carboidratos/química , Olho/química , Lectinas de Plantas , Drusas Retinianas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Metabolismo dos Carboidratos , Corioide/química , Concanavalina A/metabolismo , Histocitoquímica , Humanos , Lectinas/metabolismo , Microscopia de Fluorescência , Pessoa de Meia-Idade , Neuraminidase/metabolismo , Aglutinina de Amendoim/metabolismo , Retina/química , Aglutininas do Germe de Trigo/metabolismo
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