RESUMEN
Retinal pigment epithelium (RPE) is a monolayer of the pigmented cells that lies on the thin extracellular matrix called Bruch's membrane. This monolayer is the main component of the outer blood-retinal barrier (BRB), which plays a multifunctional role. Due to their crucial roles, the damage of this epithelium causes a wide range of diseases related to retinal degeneration including age-related macular degeneration, retinitis pigmentosa, and Stargardt disease. Unfortunately, there is presently no cure for these diseases. Clinically implantable RPE for humans is under development, and there is no practical examination platform for drug development. Here, we developed porcine Bruch's membrane-derived bioink (BM-ECM). Compared to conventional laminin, the RPE cells on BM-ECM showed enhanced functionality of RPE. Furthermore, we developed the Bruch's membrane-mimetic substrate (BMS) via the integration of BM-ECM and 3D printing technology, which revealed structure and extracellular matrix components similar to those of natural Bruch's membrane. The developed BMS facilitated the appropriate functions of RPE, including barrier and clearance functions, the secretion of anti-angiogenic growth factors, and enzyme formation for phototransduction. Moreover, it could be used as a basement frame for RPE transplantation. We established BMS using 3D printing technology to grow RPE cells with functions that could be used for an in vitro model and RPE transplantation.
Asunto(s)
Biomimética , Lámina Basal de la Coroides/citología , Degeneración Macular/patología , Impresión Tridimensional , Epitelio Pigmentado de la Retina/citología , Inhibidores de la Angiogénesis/farmacología , Animales , Adhesión Celular , Proliferación Celular , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Técnicas In Vitro , Microvellosidades , Fagocitosis , Ratas , Reología , PorcinosRESUMEN
Purpose: The purpose of this study was to determine changes in optic nerve head (ONH) morphology in seated and 6° head-down tilt (HDT) postures over a 12-hour period. Methods: Thirty eyes of 30 healthy human subjects (15 females) were included. Composite radial and circular optical coherence tomography (OCT) scans centered on the ONH, intraocular pressure (IOP), and optic nerve sheath diameter (ONSD) were acquired every two hours from 7 a.m. to 7 p.m. for both seated (n = 30) and HDT (n = 10) sessions. Global minimum rim width (BMO-MRW), total retinal thickness (TRT), retinal nerve fiber layer thickness (RNFLT), and Bruch's membrane opening (BMO) height were quantified. Results: BMO-MRW decreased an average of 9.55 ± 8.03 µm (P < 0.01) over 12 hours in a seated position (range, -26.64 to +3.36 µm), and thinning was greater in females (-13.56 vs. -5.55 µm, P = 0.004). Modest decreases in TRT from the BMO to 500 µm (P < 0.04) and RNFLT for the 2.7, 3.5, and 4.2 mm circular scans (P < 0.02) were also observed. BMO-MRW thinning was not related to changes in IOP or ONSD (P = 0.34). In HDT, IOP and ONSD increased, BMO height moved anteriorly, and BMO-MRW thinning did not occur (P > 0.1). Conclusions: The neuroretinal rim thins throughout the day in healthy individuals, and this change cannot be explained by changes in IOP or ONSD during the same time period. A HDT posture blunts the neuroretinal rim thinning observed in a seated position, suggesting a role of the translaminar pressure difference.
Asunto(s)
Inclinación de Cabeza , Disco Óptico/anatomía & histología , Sedestación , Adolescente , Adulto , Lámina Basal de la Coroides/citología , Femenino , Voluntarios Sanos , Humanos , Presión Intraocular/fisiología , Masculino , Fibras Nerviosas , Disco Óptico/diagnóstico por imagen , Nervio Óptico/diagnóstico por imagen , Células Ganglionares de la Retina/citología , Factores de Tiempo , Tomografía de Coherencia Óptica , Tonometría Ocular , Ultrasonografía , Adulto JovenRESUMEN
Retinal degenerative disorders, such as age-related macular degeneration (AMD), are one of the leading causes of blindness worldwide, however, treatments to completely stop the progression of these debilitating conditions are non-existent. Researchers require sophisticated models that can accurately represent the native structure of human retinal tissue to study these disorders. Current in vitro models used to study the retina are limited in their ability to fully recapitulate the structure and function of the retina, Bruch's membrane and the underlying choroid. Recent developments in the field of induced pluripotent stem cell technology has demonstrated the capability of retinal pigment epithelial cells to recapitulate AMD-like pathology. However, such studies utilise unsophisticated, bio-inert membranes to act as Bruch's membrane and support iPSC-derived retinal cells. This review presents a concise summary of the properties and function of the Bruch's membrane-retinal pigment epithelium complex, the initial pathogenic site of AMD as well as the current status for materials and fabrication approaches used to generate in vitro models of this complex tissue. Finally, this review explores required advances in the field of in vitro retinal modelling. STATEMENT OF SIGNIFICANCE: Retinal degenerative disorders such as age-related macular degeneration are worldwide leading causes of blindness. Previous attempts to model the Bruch's membrane-retinal pigment epithelial complex, the initial pathogenic site of age-related macular degeneration, have lacked the sophistication to elucidate valuable insights into disease mechanisms. Here we provide a detailed account of the morphological, physical and chemical properties of Bruch's membrane which may aid the fabrication of more sophisticated and physiologically accurate in vitro models of the retina, as well as various fabrication techniques to recreate this structure. This review also further highlights some recent advances in some additional challenging aspects of retinal tissue modelling including integrated fluid flow and photoreceptor alignment.
Asunto(s)
Biomimética , Lámina Basal de la Coroides/citología , Comunicación Celular , Modelos Biológicos , Retina/citología , Fenómenos Biomecánicos , HumanosRESUMEN
OBJECTIVE: To determine Bruch's membrane opening (BMO) minimum rim width (MRW) and peripapillary retinal nerve fiber layer thickness (RNFLT) measurements, acquired with optical coherence tomography (OCT) in healthy Brazilian individuals self-reported as African Descent (AD), European Descent (ED) and Mixed Descent (MD). METHODS: 260 healthy individuals (78 AD, 103 ED and 79 MD) were included in this cross-sectional study conducted at the Clinics Hospital of the University of Campinas. We obtained optic nerve head (24 radial B scans) and peripapillary retinal nerve fiber layer (3.5-mm circle scan) images in one randomly selected eye of each subject. RESULTS: After adjustment for BMO area and age, there were no significant differences in mean global MRW (P = 0.63) or RNFLT (P = 0.07) among the three groups. Regionally, there were no significant differences in either MRW or RNFLT in most sectors, except in the superonasal sector, in which both MRW and RNFLT were thinner among ED (P = 0.04, P<0.001, respectively). RNFLT was also thinner in ED in the inferonasal sector (P = 0.009). In all races, global MRW decreased and global RNFLT increased with BMO area. AD subjects had higher rates of global RNFLT decay with age (-0.32 µm/year) compared to ED and MD subjects (-0.10 µm/year and -0.08 µm/year, respectively; P = 0.01 and P = 0.02, respectively). CONCLUSIONS AND RELEVANCE: While we found no significant differences in global MRW and RNFLT among the three races, age-related thinning of the RNFLT was significantly higher in the AD subgroup, which warrants further study.
Asunto(s)
Lámina Basal de la Coroides/citología , Voluntarios Sanos , Disco Óptico/anatomía & histología , Adolescente , Adulto , Anciano , Brasil , Lámina Basal de la Coroides/diagnóstico por imagen , Femenino , Humanos , Masculino , Persona de Mediana Edad , Disco Óptico/citología , Disco Óptico/diagnóstico por imagen , Tomografía de Coherencia Óptica , Adulto JovenRESUMEN
The biological, structural and functional configuration of Bruch's membrane (BM) is significantly relevant to age-related macular degeneration (AMD) and other chorioretinal diseases, and AMD is one of the leading causes of blindness in the elderly worldwide. The configuration may worsen along with the ageing of retinal pigment epithelium and BM that finally leads to AMD. Thus, the scaffold-based tissue-engineered retina provides an innovative alternative for retinal tissue repair. The cell and material requirements for retinal repair are discussed including cell sheet engineering, decellularised membrane and tissue-engineered membranes. Further, the challenges and potential in realising a whole tissue model construct for retinal regeneration are highlighted herein. This review article provides a framework for future development of tissue-engineered retina as a preclinical model and possible treatments for AMD.
Asunto(s)
Ceguera/prevención & control , Lámina Basal de la Coroides/citología , Degeneración Macular/terapia , Retina/citología , Ingeniería de Tejidos/métodos , Ceguera/etiología , Humanos , Degeneración Macular/complicacionesRESUMEN
PURPOSE: The aim of this study was to assess the repeatability and reproducibility (R&R) of Bruch membrane opening based on minimum rim width (BMO-MRW), minimum rim area (BMO-MRA) and peripapillary retinal nerve fiber layer thickness (RNFLT) with the Spectralis optical coherence tomography (Heidelberg Engineering) for normal and glaucoma subjects. Precise measurement of these parameters can support detection of structural glaucomatous damage and progression. METHODS: This cross-sectional study included 16 healthy controls and 16 patients with glaucoma. One eye was randomly selected and included in this study. Subjects underwent 1 baseline and 3 follow-up measurements, using 3 different Spectralis optical coherence tomography devices in randomized order, each operated by a single operator. Outcome measures were global and sectorial averages of BMO-MRW and BMO-MRA, and of peripapillary RNFLT obtained from 12/14/16-degree circle scans. Coefficients of variation (COV) were calculated and a mixed-effects analysis of variance was performed to compare R&R between devices. RESULTS: COVs of global and sectorial BMO-MRW measurement under repeatability conditions ranged from 0.51% to 1.7% (normal, 0.62% to 1.3%; glaucoma, 0.64% to 2.3%). Respective COVs under reproducibility conditions ranged from 0.89% to 1.9% (normal, 0.77% to 2.8%; glaucoma, 1.1% to 2.6%). COVs of global and sectorial RNFLT measurements under repeatability conditions ranged from 0.5% to 2.8%. Respective COVs under reproducibility conditions ranged from 1.6% to 3.5%. CONCLUSIONS: For R&R, the COVs of measured parameters were by trend higher for glaucoma eyes compared with normal controls. The BMO-MRW measurement system has an excellent precision taking into account that major and minor corrections of segmentation have to be done by the examiner before evaluation.
Asunto(s)
Lámina Basal de la Coroides/diagnóstico por imagen , Glaucoma/diagnóstico , Hipertensión Ocular/diagnóstico , Disco Óptico/diagnóstico por imagen , Retina/diagnóstico por imagen , Neuronas Retinianas/citología , Tomografía de Coherencia Óptica/métodos , Lámina Basal de la Coroides/citología , Lámina Basal de la Coroides/patología , Estudios de Casos y Controles , Recuento de Células , Estudios Transversales , Progresión de la Enfermedad , Diagnóstico Precoz , Femenino , Glaucoma/patología , Humanos , Presión Intraocular , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Fibras Nerviosas/patología , Fibras Nerviosas/ultraestructura , Hipertensión Ocular/patología , Disco Óptico/citología , Disco Óptico/patología , Tamaño de los Órganos , Reproducibilidad de los Resultados , Retina/citología , Retina/patología , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/patología , Neuronas Retinianas/patologíaRESUMEN
Purpose: To identify determinants of Bruch's membrane opening (BMO), and BMO-minimum rim width (BMO-MRW) and circumpapillary retinal nerve fiber layer thickness (RNFLT) centered on BMO center and characterize these parameters in a normal Japanese population. Methods: Spectral-domain optical coherence tomography images of optic nerve head and circumpapillary and macular retina were obtained in 258 eyes of 258 normal Japanese with mean (standard deviation) age of 51.7 (18.2) years. BMO area, BMO-MRW, RNFLT (measured with a 3.5-mm-diameter circle scan) were all acquired and analyzed relative to the eye-specific fovea to BMO (FoBMO) axis. One randomly selected eye of each subject was analyzed. Multiple regression analysis was used to identify determinants to the parameters. Results: BMO area, global BMO-MRW, RNFLT, and FoBMO angle averaged 2.06 (0.45) mm2, 305.5 (50.0) µm, 101.8 (9.6) µm, and -7.8° (3.8°), respectively. There was a modest correlation between global BMO-MRW and RNFLT (r = 0.337; P < 0.001), while the sectorwise correlations were highest in the superior-temporal sector (r = 0.500; P < 0.001) and lowest in the nasal sector (r = 0.117; P = 0.063). Global BMO-MRW and RNFLT declined with age at -1.04 µm/y (P < 0.001) and -0.12 µm/y (P = 0.001), and the former correlated negatively (P = 0.001) and the latter positively (P < 0.001) with BMO area after adjustment for other factors (R2 = 0.191 and 0.272, respectively). BMO area correlated positively with axial length (P = 0.023) and negatively with age (P < 0.001) (R2 = 0.157). Conclusions: BMO-MRW and RNFLT declined with age with a difference between them in their relationship to BMO area. BMO area positively correlated with axial length and negatively with age.
Asunto(s)
Lámina Basal de la Coroides/citología , Disco Óptico/citología , Células Ganglionares de la Retina/citología , Tomografía de Coherencia Óptica/métodos , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Fibras Nerviosas , Valores de ReferenciaRESUMEN
The chorioretinal junction comprises the retinal pigment epithelium, Bruch's membrane (BM), and adjacent choroidal capillaries. Its significance lies in its ability to support the retina mechanically and metabolically. The aim of this cross-sectional study was to record the senescent changes affecting all the constituents of the chorioretinal junction in 40 histological specimens across the whole spectrum of the adult age range. This study included light microscopy, with hematoxylin and eosin and PAS stains, and fluorescent microscopy. Immunohistochemistry was done using antibodies against neurofilament, synaptophysin, S-100, and collagen IV. The descriptive microanatomy was corroborated by morphometry. The amount of melanin and lipofuscin granule and drusens were noted. The ratio of thickness of BM to capillary diameter reduced from 1:6 or less in the 2nd decade to 1:3 in the 10th decade. Complete hyalinization of intercapillary pillars was seen in the 10th decade. The accumulation of lipofuscin with age was documented with the diminution in the size of epithelial cells. The subepithelial accumulation of drusen was first noted in the specimen from the late 60s. We have described all senescent changes in the chorioretinal junction chronologically. Similar changes are found in a more pronounced form in age-related macular degeneration. These data might serve as a reference baseline for clinicians and pathologists.
Asunto(s)
Envejecimiento/metabolismo , Coroides/citología , Retina/citología , Adulto , Anciano , Anciano de 80 o más Años , Lámina Basal de la Coroides/citología , Lámina Basal de la Coroides/metabolismo , Coroides/metabolismo , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Adulto JovenRESUMEN
Transplanted retinal pigment epithelium (RPE) cells hold promise for treatment of age-related macular degeneration (AMD) and Stargardt disease (SD), but it is conceivable that the degenerated host Bruch's membrane (BM) as a natural substrate for RPE might not optimally support transplanted cell survival with correct cellular organization. We fabricated novel ultrathin three-dimensional (3-D) nanofibrous membranes from collagen type I and poly(lactic-co-glycolic acid) (PLGA) by an advanced clinical-grade needle-free electrospinning process. The nanofibrillar 3-D networks closely mimicked the fibrillar architecture of the native inner collagenous layer of human BM. Human RPE cells grown on our nanofibrous membranes bore a striking resemblance to native human RPE. They exhibited a correctly orientated monolayer with a polygonal cell shape and abundant sheet-like microvilli on their apical surfaces. RPE cells built tight junctions and expressed RPE65 protein. Flat 2-D PLGA film and cover glass as controls delivered inferior RPE layers. Our nanofibrous membranes may imitate the natural BM to such extent that they allow for the engineering of an in vivo-like human RPE monolayer that maintains the natural biofunctional characteristics. Such ultrathin membranes may provide a promising vehicle for a functional RPE cell monolayer implantation in the subretinal space in patients with AMD or SD.
Asunto(s)
Lámina Basal de la Coroides/citología , Células Epiteliales/citología , Nanofibras/química , Epitelio Pigmentado de la Retina/citología , Ingeniería de Tejidos/métodos , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Colágeno/metabolismo , Colágeno/ultraestructura , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Humanos , Inmunohistoquímica , Ácido Láctico/farmacología , Nanofibras/ultraestructura , Fagocitosis/efectos de los fármacos , Ácido Poliglicólico/farmacología , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Proteína de la Zonula Occludens-1/metabolismo , cis-trans-Isomerasas/metabolismoRESUMEN
There is a mutualistic symbiotic relationship between the components of the photoreceptor/retinal pigment epithelium (RPE)/Bruch's membrane (BrMb)/choriocapillaris (CC) complex that is lost in AMD. Which component in the photoreceptor/RPE/BrMb/CC complex is affected first appears to depend on the type of AMD. In atrophic AMD (~85-90% of cases), it appears that large confluent drusen formation and hyperpigmentation (presumably dysfunction in RPE) are the initial insult and the resorption of these drusen and loss of RPE (hypopigmentation) can be predictive for progression of geographic atrophy (GA). The death and dysfunction of photoreceptors and CC appear to be secondary events to loss in RPE. In neovascular AMD (~10-15% of cases), the loss of choroidal vasculature may be the initial insult to the complex. Loss of CC with an intact RPE monolayer in wet AMD has been observed. This may be due to reduction in blood supply because of large vessel stenosis. Furthermore, the environment of the CC, basement membrane and intercapillary septa, is a proinflammatory milieu with accumulation of complement components as well as proinflammatory molecules like CRP during AMD. In this toxic milieu, CC die or become dysfunction making adjacent RPE hypoxic. These hypoxic cells then produce angiogenic substances like VEGF that stimulate growth of new vessels from CC, resulting in choroidal neovascularization (CNV). The loss of CC might also be a stimulus for drusen formation since the disposal system for retinal debris and exocytosed material from RPE would be limited. Ultimately, the photoreceptors die of lack of nutrients, leakage of serum components from the neovascularization, and scar formation. Therefore, the mutualistic symbiotic relationship within the photoreceptor/RPE/BrMb/CC complex is lost in both forms of AMD. Loss of this functionally integrated relationship results in death and dysfunction of all of the components in the complex.
Asunto(s)
Degeneración Macular/etiología , Degeneración Macular/metabolismo , Animales , Lámina Basal de la Coroides/citología , Lámina Basal de la Coroides/patología , Coroides/citología , Coroides/patología , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Células Fotorreceptoras/citología , Células Fotorreceptoras/patología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/patologíaRESUMEN
PURPOSE: Neovascular AMD involves the activation of choroidal endothelial cells to increase their inflammatory and angiogenic behaviors. Elastin derived peptides (EDPs) can elicit some of these phenotypic changes in endothelial cells. This investigation was performed to follow up on those findings by determining a receptor for these peptides in the human eye as well as evaluating the effects of elevated EDPs on choroidal cells in vitro and in vivo. METHODS: The expression of elastin receptor genes including GLB1 was analyzed using reverse transcription PCR. Migration of choroidal endothelial cells was quantified in the presence of inhibitors to different EDP binding proteins. C57BL6 mice were injected with EDPs and studied by electroretinography, transmission electron microscopy, and microarray analysis. RESULTS: An alternatively spliced form of beta-galactosidase (GLB1) is present on human choroidal endothelial cells and acts as a receptor for EDPs. Elevated levels of circulating EDPs do not affect retinal function in the mouse, but do increase the expression and deposition of collagen IV in the RPE/choroid complex. CONCLUSIONS: EDPs may play a role in neovascular AMD by binding to and inducing neovascular phenotypes in choroidal endothelial cells through their receptor, GLB1. These peptides also cause an increased mRNA expression and deposition of collagen IV in the RPE/choroid, which may alter diffusion properties between the retina and choriocapillaris.
Asunto(s)
Lámina Basal de la Coroides/citología , Coroides/patología , Elastina/farmacología , Células Endoteliales/patología , beta-Galactosidasa/metabolismo , Empalme Alternativo , Animales , Catepsina A/genética , Catepsina A/metabolismo , Línea Celular , Ensayos de Migración Celular , Movimiento Celular , Coroides/efectos de los fármacos , Coroides/metabolismo , Difusión , Electrorretinografía , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neuraminidasa/genética , Neuraminidasa/metabolismo , Péptidos/farmacología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retina/metabolismo , Retina/patología , Retina/ultraestructura , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , beta-Galactosidasa/genéticaRESUMEN
PURPOSE: To compare RPE derived from human embryonic stem cells (hES-RPE) and fetal RPE (fRPE) behavior on human Bruch's membrane (BM) from aged and AMD donors. METHODS: hES-RPE of 3 degrees of pigmentation and fRPE were cultured on BM explants. Explants were assessed by light, confocal, and scanning electron microscopy. Integrin mRNA levels were determined by real-time polymerase chain reaction studies. Secreted proteins in media were analyzed by multiplex protein analysis after 48-hour exposure at culture day 21. RESULTS: hES-RPE showed impaired initial attachment compared to fRPE; pigmented hES-RPE showed nuclear densities similar to fRPE at day 21. At days 3 and 7, hES-RPE resurfaced BM to a limited degree, showed little proliferation (Ki-67), and partial retention of RPE markers (MITF, cytokeratin, and CRALBP). TUNEL-positive nuclei were abundant at day 3. fRPE exhibited substantial BM resurfacing at day 3 with decreased resurfacing at later times. Most fRPE retained RPE markers. Ki-67-positive nuclei decreased with time in culture. TUNEL staining was variable. Increased integrin mRNA expression did not appear to affect cell survival at day 21. hES-RPE and fRPE protein secretion was similar on equatorial BM except for higher levels of nerve growth factor and thrombospondin-2 (TSP2) by hES-RPE. On submacular BM, fRPE secreted more vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor, and platelet-derived growth factor; hES-RPE secreted more TSP2. CONCLUSIONS: Although pigmented hES-RPE and fRPE resurfaced aged and AMD BM to a similar, limited degree at day 21, cell behavior at earlier times was markedly dissimilar. Differences in protein secretion may indicate that hES-RPE may not function identically to native RPE after seeding on aged or AMD BM.
Asunto(s)
Envejecimiento/fisiología , Lámina Basal de la Coroides/citología , Células Madre Embrionarias/citología , Células Madre Fetales/citología , Epitelio Pigmentado de la Retina/citología , Anciano , Anciano de 80 o más Años , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Etiquetado Corte-Fin in Situ , Integrinas/genética , Queratinas/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Degeneración Macular/patología , Masculino , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Microscopía Confocal , Microscopía Electrónica de Rastreo , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , ARN Mensajero/metabolismo , Epitelio Pigmentado de la Retina/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A quantitative proteomics analysis of the macular Bruch membrane/choroid complex was pursued for insights into the molecular mechanisms of age-related macular degeneration (AMD). Protein in trephine samples from the macular region of 10 early/mid-stage dry AMD, six advanced dry AMD, eight wet AMD, and 25 normal control post-mortem eyes was analyzed by LC MS/MS iTRAQ (isobaric tags for relative and absolute quantitation) technology. A total of 901 proteins was quantified, including 556 proteins from > or =3 AMD samples. Most proteins differed little in amount between AMD and control samples and therefore reflect the proteome of normal macular tissues of average age 81. A total of 56 proteins were found to be elevated and 43 were found to be reduced in AMD tissues relative to controls. Analysis by category of disease progression revealed up to 16 proteins elevated or decreased in each category. About 60% of the elevated proteins are involved in immune response and host defense, including many complement proteins and damage-associated molecular pattern proteins such as alpha-defensins 1-3, protein S100s, crystallins, histones, and galectin-3. Four retinoid processing proteins were elevated only in early/mid-stage AMD, supporting a role for retinoids in AMD initiation. Proteins uniquely decreased in early/mid-stage AMD implicate hematologic malfunctions and weakened extracellular matrix integrity and cellular interactions. Galectin-3, a receptor for advanced glycation end products, was the most significantly elevated protein in advanced dry AMD, supporting a role for advanced glycation end products in dry AMD progression. The results endorse inflammatory processes in both early and advanced AMD pathology, implicate different pathways of progression to advanced dry and wet AMD, and provide a new database for hypothesis-driven and discovery-based studies of AMD.
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Lámina Basal de la Coroides/metabolismo , Lámina Basal de la Coroides/patología , Proteínas del Ojo/metabolismo , Degeneración Macular/metabolismo , Degeneración Macular/patología , Proteómica/métodos , Anciano , Anciano de 80 o más Años , Western Blotting , Lámina Basal de la Coroides/citología , Progresión de la Enfermedad , Femenino , Humanos , Degeneración Macular/clasificación , Masculino , Proteoma/metabolismoRESUMEN
Current efforts to reverse loss of visual function due to Age-related Macular Degeneration point to the restoration of the Retinal Pigment Epithelial (RPE) layer. Restoration of the RPE layer involves replacing lost RPE cells as well as addressing the degeneration of the underlying Bruch's membrane (BM). To advance the potential of using donor BM, we present a strategy to achieve specific and controllable modification of the inner collagenous layer (ICL) of the Bruch's membrane. In particular, interaction between a collagen binding peptide (CBP) sequence with exposed collagen fibers on the ICL surface is utilized to anchor bioactive molecules. Here, a cell-adhesion sequence is added to the collagen binding sequence to promote attachment and survival of ARPE-19. First, the binding specificity of the CBP sequence is verified with a fluorescent binding assay. Subsequently, the effect of modification using the peptide is studied qualitatively using confocal fluorescent imaging and quantitatively through a cell proliferation assay. Results of these experiments indicate that the peptide sequence binds specifically to collagen fibers. Additionally, modification using the peptide enhanced cell adhesion, allowing large uniform cell networks to be formed on the surface. Furthermore, modification with the peptide also delayed the onset of apoptosis on adherent cells.
Asunto(s)
Lámina Basal de la Coroides/metabolismo , Adhesión Celular , Colágeno/metabolismo , Péptidos/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Análisis de Varianza , Animales , Apoptosis , Lámina Basal de la Coroides/citología , Compuestos de Dansilo/metabolismo , Microscopía Fluorescente , Microscopía de Contraste de Fase , Epitelio Pigmentado de la Retina/citología , PorcinosRESUMEN
BACKGROUND: Translocation of an autologous retinal pigment epithelium (RPE) sheet under the macula is currently under investigation as a treatment for exudative AMD. Excimer laser-assisted RPE sheet translocation (EST) employs intraocular excimer ablation of excess graft choroidal tissue as a measure to enhance RPE sheet functionality. This study assessed potential adverse effects of excimer irradiation on RPE cells in vitro. METHODS: Human RPE cells (ARPE-19) received 308 nm XeCl excimer laser treatment or 311-312 nm UV-B irradiation. Cell death was visualised with Trypan Blue and quantified by LDH release assay. Apoptosis was detected by DNA fragmentation assay. RESULTS: Laser treatment of 0.175-0.25 J/cm(2) resulted in delayed cell death within 48 h. Time course and dose response paralleled the effect of UV-B irradiation. Cytotoxicity was mediated by apoptosis. Human choroid/Bruch membrane tissue sheets covering the cells during laser irradiation reduced cytotoxicity by 87-95%. CONCLUSION: Cultured human RPE cells are susceptible to apoptotic cell death induced by 308 nm excimer laser irradiation. Absorption by choroid/Bruch membrane tissue can largely prevent the cytotoxic effect. In clinical application, the residual adverse effect of laser ablation on graft RPE cell viability needs to be outweighed by potential advantageous effects on graft survival and functionality to allow for a sensible application of excimer ablation in RPE translocation surgery.
Asunto(s)
Láseres de Excímeros/efectos adversos , Degeneración Macular/cirugía , Epitelio Pigmentado de la Retina/citología , Lámina Basal de la Coroides/citología , Muerte Celular/fisiología , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Coroides/citología , Supervivencia de Injerto/fisiología , Humanos , Degeneración Macular/metabolismo , Epitelio Pigmentado de la Retina/efectos de la radiación , Epitelio Pigmentado de la Retina/cirugíaRESUMEN
To determine the effects of extracellular matrix and neighboring cells on the differentiation of human embryonic stem cells (hESC) into progenitors of retinal cells and/or retinal pigment epithelium (RPE). HESC were cultured on mouse PA6 stromal cells for approximately 2weeks to obtain neural progenitors. To induce photoreceptor marker expression, the neural progenitors were cultured on a confluent monolayer of ARPE19 or on laminin-coated dishes. To induce RPE markers, the neural progenitors were seeded onto human Bruch's membrane or Matrigel. Cells were examined morphologically and stained with different RPE or neural progenitor markers. Microarray techniques were used to compare the gene expression profiles of hESC cultured on mouse fibroblasts or neural progenitors on PA6 cells to the transcriptome of the adult neural retina and RPE. HESC cultured on PA6 cells expressed neural progenitor markers beta-tubulin III, PAX6, neural filament, GFAP and vimentin. Culturing these neural progenitors on confluent ARPE19 monolayer induced expression of the photoreceptor progenitor cell marker CRX; culturing neural progenitors on laminin substrates induced a neuronal phenotype with neurite formation. Neural progenitors expressed the RPE marker ZO-1 after culturing on Matrigel-coated dishes and the RPE marker Bestrophin after culturing on human Bruch's membrane explants. Hierarchical clustering analysis of samples suggested that when cultured on PA6 stromal cells hESC exhibited genetic characteristics towards differentiating into neural retina. Microarray analysis showed that after culturing on PA6 cells, stem cells expressed 117 new genes; among these there were 22 genes present in neural retina or RPE cells. The functions of these genes were highly related to cell proliferation, nervous system development and cell adhesion. HESC can be induced to differentiate into neural progenitors after culturing on PA6 cells. These neural progenitors can express RPE markers when cultured on Bruch's membrane or Matrigel, or photoreceptor markers when cultured on confluent ARPE19 or laminin. Additional studies are required to assess the function of hESC induced to express retinal or RPE markers prior to successful intraocular transplantation into animal models of retinal degeneration.
Asunto(s)
Células Madre Embrionarias/citología , Matriz Extracelular/fisiología , Epitelio Pigmentado Ocular/citología , Retina/citología , Animales , Lámina Basal de la Coroides/citología , Comunicación Celular/fisiología , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Madre Embrionarias/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Epitelio Pigmentado Ocular/metabolismo , Retina/metabolismo , Células del Estroma/citologíaRESUMEN
The purpose of this study was to examine the change in integrin expression in adult human retinal pigment epithelium (RPE) after culturing and to characterize the role of integrins in RPE adhesion to aged submacular human Bruch's membrane. Expression of alpha integrin subunits 1 through 6 in adult RPE cells, cultured or uncultured, was examined by reverse transcription/real-time polymerase chain reaction (PCR) and Western blotting. RPE was cultured on bovine corneal endothelial cell-secreted extracellular matrix (BCE-ECM). The role of alpha integrin subunits in RPE attachment was examined by immunofluorescent localization of these subunits at sites of focal adhesions in cultured adult RPE attached to laminin or collagen-I-coated culture dishes. Additionally, the effect of function-blocking antibodies to alpha integrin subunits on RPE attachment to laminin, collagen I, and aged submacular human Bruch's membrane was determined. Cultured adult RPE had increased expression of alpha1-5 integrin subunits by PCR compared to uncultured RPE. Western blots showed that alpha2, 3, and 5 subunit levels were low or absent in uncultured adult RPE. Cultured adult RPE had a substantially higher expression of these integrins. Alpha 1-3 subunits co-localized with phosphorylated focal adhesion kinase (FAK) at focal adhesions in RPE cells spread on laminin. Only alpha2 and alpha3 co-localized with phosphorylated FAK in focal adhesions of RPE on collagen I. Using function blocking antibodies, blocking alpha1 subunit singly or in combination with alpha2 and/or alpha3 significantly decreased RPE adhesion to laminin. Blocking alpha1 and alpha2 or blocking alpha1, alpha2, and alpha3 subunits significantly decreased RPE adhesion to collagen I. Compared to controls, significantly fewer RPE cells were able to spread on aged submacular human Bruch's membrane when alpha1-6 integrin subunits were blocked. These results indicate that alpha 1-5 subunits that are upregulated by culturing on BCE-ECM are necessary for RPE attachment to aged submacular human Bruch's membrane. Relative lack of these integrin subunits in uncultured adult RPE may be responsible for poor resurfacing of aged submacular human Bruch's membrane by these cells.
Asunto(s)
Lámina Basal de la Coroides/metabolismo , Cadenas alfa de Integrinas/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Envejecimiento/patología , Western Blotting/métodos , Lámina Basal de la Coroides/citología , Adhesión Celular/fisiología , Células Cultivadas , Colágeno Tipo I/metabolismo , Humanos , Cadenas alfa de Integrinas/genética , Cadenas alfa de Integrinas/fisiología , Laminina/metabolismo , Microscopía Fluorescente/métodos , Persona de Mediana Edad , Epitelio Pigmentado Ocular/citología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The outer blood-retinal barrier is composed of a monolayer of retinal pigment epithelium, Bruch's membrane and the choriocapillaris which is fenestrated. Endothelial proliferation and breaching of Bruch's membrane leads to the neovascular form of age-related macula degeneration (ARMD). The aim of this study was to generate an in vitro model that mimics more faithfully the phenotype of the choriocapillaris and the trilayer architecture in vitro. A trilayer culture model was generated with retinal pigment epithelium (ARPE-19) cell cultures on the epithelial surface of amniotic membrane and with human umbilical vein-derived endothelial cells on the other surface. A control model for the effect of retinal pigment epithelium on endothelial changes was generated with corneal epithelial cells replacing the ARPE-19. Both human umbilical vein-derived endothelial and ARPE-19 cells formed confluent monolayers on respective surfaces of the amnion. The human umbilical vein-derived endothelial cells in the trilayer became fenestrated when co-cultured with the ARPE-19 cells, but not with corneal epithelial cells, or when grown as monolayers on the amnion, showing a loss of fidelity of origin in the presence of ARPE-19 cells. These cells also revealed VE-cadherin and ZO-1 at cell-cell contacts from 24 h in the trilayer. The tight junctional molecules, occludin and ZO-1, were localized to cell-cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. Permeability of the trilayer was tested by using fluorescein and fluorescein-conjugated tracers under flow. At 72 h the trilayer severely restricted transfer of sodium fluorescein (NaF) (ten-fold reduction) whilst transfer of a 4 kDa FITC-conjugated dextran was virtually occluded, confirming a restrictive barrier. Ultrastructural studies showed the retinal pigment epithelium monolayer was polarized with microvilli present on the apical surface. Paracellular clefts showed numerous tight junctional-like appositions, similar to that seen on amnion alone. This study demonstrates that ARPE-19 and human umbilical vein-derived endothelial cells can be co-cultured on the amniotic membrane and that the resultant cross-talk leads to formation of a fenestrated endothelium, whilst maintaining a polarized restrictive epithelial layer. The fenestrated endothelial phenotype achieved in this human in vitro trilayer model is a first and offers an outer-retinal barrier which approaches the in vivo state and has potential for studies into induced junctional disruption, endothelial proliferation and migration: features of ARMD.
Asunto(s)
Barrera Hematorretinal/citología , Amnios/citología , Transporte Biológico , Lámina Basal de la Coroides/citología , Permeabilidad Capilar , Células Cultivadas , Técnicas de Cocultivo , Células Epiteliales/citología , Humanos , Inmunohistoquímica , Microscopía Electrónica , Modelos Biológicos , Epitelio Pigmentado Ocular/citología , Uniones Estrechas , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
PURPOSE: Human retina and retinal pigment epithelium (RPE) express a relatively abundant mRNA that encodes an extraneous splice isoform of the RPE retinal G protein-coupled receptor (RGR) opsin. In this study, we investigate this exon-skipping RGR splice isoform (RGR-d) in separated neural retina and RPE cells of human donors of various ages. METHODS: We used mass spectrometry, sensitive western blot assay, immunohistochemical localization and real-time RT-PCR to analyze RGR-d. RESULTS: Western blot assay detected the RGR-d protein in the neural retina of all donors analyzed. Mass spectrometric analysis of the immunoreactive proteins independently confirmed the presence of RGR-d. In contrast, RGR-d protein in the RPE of most donors was barely detectable by western blot assay, even though expression of RGR-d mRNA was confirmed by amplification of RGR-d transcripts in both the RPE and neural retina. Quantitative real-time RT-PCR assays showed that RGR-d/RGR mRNA transcript ratios were about 0.17 and about 0.33 in the RPE and neural retina, respectively. Immunohistochemical localization studies revealed that the RGR-d epitope was present near the basal boundary of RPE cells and primarily in the extracellular areas of Bruch's membrane, adjacent choriocapillaris, and intercapillary region of both young and older donors. Positive immunostaining was seen in the drusen of older individuals. CONCLUSIONS: The RGR-d protein is a common mutant form of human RGR that can be identified in donor eyes by mass spectrometry. These results indicate that after RGR-d is synthesized, the RGR-d epitope is released at the basal surface of the RPE and deposited into Bruch's membrane in human eyes throughout adult life.
Asunto(s)
Empalme Alternativo/genética , Lámina Basal de la Coroides/metabolismo , Exones/genética , Proteínas del Ojo/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adolescente , Adulto , Anciano , Secuencia de Aminoácidos , Lámina Basal de la Coroides/citología , ADN Complementario/metabolismo , Epítopos/química , Proteínas del Ojo/química , Proteínas del Ojo/genética , Femenino , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Datos de Secuencia Molecular , Epitelio Pigmentado Ocular/citología , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The morphology of the retinal pigment epithelium (RPE) and closely associated Bruch's membrane and choriocapillaris was investigated by light and transmission electron microscopy in the camel (Camelus dromedarius). The study showed that RPE is composed of a single layer of hexanocuboidal cells that were joined laterally by a series of apically located tight junctions. In addition, adjacent from internal side of cell membrane at the level of tight junctions, an undefined structure which resembled the myofibrillar organization of skeletal muscles in appearance was located. These cells displayed numerous short basal infoldings and abundant thin apical processes which enclosed the rod outer segments. The epithelial cell nuclei were large, vesicular and eccentrically located. Within the epithelial cells, smooth endoplasmic reticulum was very abundant, while rough endoplasmic reticulum was present only in small amounts. Polysomes were also numerous and the mitochondria often displayed a ring-shaped structure. Lipofuscin granules were plentiful in all locations. Bruch's membrane (complexus basalis) was typically pentalaminate throughout the retina. The endothelium of the choriocapillaris facing Bruch's membrane was extremely thin and heavily fenestrated. These fenestrations displayed typical single-layered diaphragm as noted in most species.