RESUMEN
The choriocapillaris is the source of nutrients and oxygen for photoreceptors, which consume more oxygen per gram of tissue than any other cell in the body. The purpose of this study was to evaluate and compare the ultrastructure of the choriocapillaris and its transport systems in patients with and without age-related macular degeneration (AMD). Ultrastructural changes were also evaluated in subjects that were homozygous for polymorphisms in high risk CFH alleles (Pure 1) only or homozygous only for high risk ARMS2/HTRA1 (Pure 10) alleles. Tissue samples were obtained from the macular region of forty male (nâ¯=â¯24) and female (nâ¯=â¯16) donor eyes and prepared for ultrastructural studies with transmission electron microscopy (TEM). The average age of the aged donors was 74⯱â¯7.2 (nâ¯=â¯30) and the young donors 31.7⯱â¯11.25 (nâ¯=â¯10). There was no significant difference in average ages between the adult groups. TEM images of the capillaries in the choriocapillaris (CC) were taken at 4,000X and 25,000X and used to measure the area of endothelial cell somas, the number of fenestrations, and area of caveolae within the endothelial cells per length of Bruchs membrane (BrMb). The Student t-test and Wilcoxon sum rank test were used to determine significant differences. There was no significant difference between young subjects and aged controls in any of the morphological criteria assessed. There was a significant decrease in the number of fenestrations/mm of BrMb in atrophic areas of GA eyes (pâ¯=â¯0.007) when compared with aged control eyes. A significant increase was found in the caveolae area as a percent of the endothelial cell soma of capillaries from GA subjects as compared with the controls (pâ¯=â¯0.03). Loss of capillary segments in choriocapillaris was also evident, especially in areas of geographic atrophy and CNV. In eyes from patients with sequence variations, the capillary endothelial cells often appeared degenerative and exhibited atypical fenestrations and pericytes covering the blood vessels. Subjects that were homozygous for polymorphisms in high risk CFH alleles only had more fenestrations/mm of BrMb than subjects that were homozygous only for high risk ARMS2/HTRA1 alleles (pâ¯=â¯0.04), while the latter had greater caveolae area/endothelial cell area than the former (pâ¯=â¯0.007). This study demonstrated an attenuation of CC and a significant decline in the two major transport systems in CC endothelial cells in AMD. This may contribute to drusen deposition, nutrient transport, and vision loss in AMD subjects.
Asunto(s)
Coroides/ultraestructura , Oxígeno/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Degeneración Macular Húmeda/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Coroides/metabolismo , Femenino , Humanos , Transporte Iónico , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Epitelio Pigmentado de la Retina/ultraestructura , Adulto JovenRESUMEN
During organogenesis, PAX6 is required for establishment of various progenitor subtypes within the central nervous system, eye and pancreas. PAX6 expression is maintained in a variety of cell types within each organ, although its role in each lineage and how it acquires cell-specific activity remain elusive. Herein, we aimed to determine the roles and the hierarchical organization of the PAX6-dependent gene regulatory network during the differentiation of the retinal pigmented epithelium (RPE). Somatic mutagenesis of Pax6 in the differentiating RPE revealed that PAX6 functions in a feed-forward regulatory loop with MITF during onset of melanogenesis. PAX6 both controls the expression of an RPE isoform of Mitf and synergizes with MITF to activate expression of genes involved in pigment biogenesis. This study exemplifies how one kernel gene pivotal in organ formation accomplishes a lineage-specific role during terminal differentiation of a single lineage.
Asunto(s)
Diferenciación Celular/genética , Proteínas del Ojo/biosíntesis , Proteínas de Homeodominio/biosíntesis , Factor de Transcripción Asociado a Microftalmía/genética , Organogénesis/genética , Factores de Transcripción Paired Box/biosíntesis , Proteínas Represoras/biosíntesis , Animales , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Ratones , Factor de Transcripción Asociado a Microftalmía/biosíntesis , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Pigmentación/genética , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Epitelio Pigmentado de la Retina/crecimiento & desarrollo , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
The retinal pigmented epithelium (RPE) is critically important to retinal homeostasis, in part due to its very active processes of phagocytosis and autophagy. Both of these processes depend upon the normal functioning of lysosomes, organelles which must fuse with (auto)phagosomes to deliver the hydrolases that effect degradation of cargo. It has become clear that signaling through mTOR complex 1 (mTORC1), is very important in the regulation of lysosomal function. This signaling pathway is becoming a target for therapeutic intervention in diseases, including age-related macular degeneration (AMD), where lysosomal function is defective. In addition, our laboratory has been studying animal models in which the gene (Cryba1) for ßA3/A1-crystallin is deficient. These animals exhibit impaired lysosomal clearance in the RPE and pathological signs that are similar to some of those seen in AMD patients. The data demonstrate that ßA3/A1-crystallin localizes to lysosomes in the RPE and that it is a binding partner of V-ATPase, the proton pump that acidifies the lysosomal lumen. This suggests that ßA3/A1-crystallin may also be a potential target for therapeutic intervention in AMD. In this review, we focus on effector molecules that impact the lysosomal-autophagic pathway in RPE cells.
Asunto(s)
Autofagia/fisiología , Lisosomas/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Animales , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos/fisiología , Biogénesis de Organelos , Serina-Treonina Quinasas TOR/fisiologíaRESUMEN
BACKGROUND: The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSCs for treating ischemic retinopathies, we generated VPs from a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)-derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model. METHODS AND RESULTS: VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing endothelial/pericytic markers identified a CD31(+)CD146(+) VP population with high vascular potency. Episomal CB-induced pluripotent stem cells (iPSCs) generated these VPs with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to fibroblast-iPSC-VPs, CB-iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VPs from human embryonic stem cells, CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia-reperfusion-injured adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell- and CB-iPSC-derived VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days. CONCLUSIONS: VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.
Asunto(s)
Células Madre Embrionarias/citología , Sangre Fetal/citología , Células Madre Pluripotentes/citología , Daño por Reperfusión/terapia , Enfermedades de la Retina/terapia , Trasplante de Células Madre/métodos , Animales , Capilares/citología , Senescencia Celular , Daño del ADN , Modelos Animales de Enfermedad , Fibroblastos/citología , Supervivencia de Injerto , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Regeneración , Daño por Reperfusión/patología , Enfermedades de la Retina/patología , TranscriptomaRESUMEN
Fuchs endothelial corneal dystrophy (FECD) is a leading indication for corneal transplantation. FECD is characterized by progressive alterations in endothelial cell morphology, excrescences (guttae) and thickening of the endothelial basement membrane and cell death. Ultimately, these changes lead to corneal edema and vision loss. Due to the lack of vision loss in early disease stages and the decades long disease course, early pathophysiology in FECD is virtually unknown as studies of pathologic tissues have been limited to end-stage tissues obtained at transplant. The first genetic defect shown to cause FECD was a point mutation causing a glutamine to lysine substitution at amino acid position 455 (Q455K) in the alpha 2 collagen 8 gene (COL8A2) which results in an early onset form of the disease. Homozygous mutant knock-in mice with this mutation (Col8a2(Q455K/Q455K)) show features strikingly similar to human disease, including progressive alterations in endothelial cell morphology, cell loss and basement membrane guttae. Ultrastructural analysis shows the predominant defect as dilated endoplasmic reticulum (ER), suggesting ER stress and unfolded protein response (UPR) activation. Immunohistochemistry, western blotting, quantitative reverse transcriptase polymerase chain reaction and terminal deoxynucleotidyl transferase 2-deoxyuridine, 5-triphosphate nick end-labeling analyses support UPR activation and UPR-associated apoptosis in the Col8a2(Q455K/Q455K) mutant corneal endothelium. This study confirms the Q455K substitution in the COL8A2 gene as being sufficient to cause FECD in the first mouse model of this disease and supports the role of the UPR and UPR-associated apoptosis in the pathogenesis of FECD caused by COL8A2 mutations.
Asunto(s)
Apoptosis , Colágeno Tipo VIII/genética , Modelos Animales de Enfermedad , Endotelio Corneal/metabolismo , Distrofia Endotelial de Fuchs/patología , Animales , Endotelio Corneal/patología , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/metabolismo , Ratones , Ratones Transgénicos , Microscopía Electrónica , Mutación , Desnaturalización ProteicaRESUMEN
Purpose: Geographic atrophy (GA) is an advanced form of dry age-related macular degeneration with multifactorial etiology and no well-established treatment. A model recapitulating the hallmarks would serve as a key to understanding the underlying pathologic mechanisms better. In this report, we further characterized our previously reported subretinal sodium iodate model of GA. Methods: Retinal degeneration was induced in rats (6-8 weeks old) by subretinal injections of NaIO3 as described previously. Animals were sacrificed at 3, 8 and 12 weeks after injection and eyes were fixed or cryopreserved. Some choroids were processed as flatmounts while other eyes were cryopreserved, sectioned, and immunolabeled with a panel of antibodies. Finally, some eyes were prepared for transmission electron microscopic (TEM) analysis. Results: NaIO3 subretinal injection resulted in a well-defined focal area of retinal pigment epithelium (RPE) degeneration surrounded by viable RPE. These atrophic lesions expanded over time. RPE morphologic changes at the border consisted of hypertrophy, multilayering, and the possible development of a migrating phenotype. Immunostaining of retinal sections demonstrated external limiting membrane descent, outer retinal tubulation (ORT), and extension of Müller cells toward RPE forming a glial membrane in the subretinal space of the atrophic area. TEM findings demonstrated RPE autophagy, cellular constituents of ORT, glial membranes, basal laminar deposits, and defects in Bruch's membrane. Conclusions: In this study, we showed pathologic features of a rodent model resembling human GA in a temporal order through histology, immunofluorescence, and TEM analysis and gained insights into the cellular and subcellular levels of the GA-like phenotypes. Translational Relevance: Despite its acute nature, the expansion of atrophy and the GA-like border in this rat model makes it ideal for studying disease progression and provides a treatment window to test potential therapeutics for GA.
Asunto(s)
Atrofia Geográfica , Degeneración Retiniana , Humanos , Ratas , Animales , Retina , Epitelio Pigmentado de la Retina/patología , Yodatos , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/patologíaRESUMEN
Phagocytosis of the shed outer segment discs of photoreceptors is a major function of the retinal pigmented epithelium (RPE). We demonstrate for the first time that ßA3/A1-crystallin, a major structural protein of the ocular lens, is expressed in RPE cells. Further, by utilizing the Nuc1 rat, in which the ßA3/A1-crystallin gene is mutated, we show that this protein is required by RPE cells for proper degradation of outer segment discs that have been internalized in phagosomes. We also demonstrate that in wild-type RPE, ßA3/A1-crystallin is localized to the lysosomes. However, in the Nuc1 RPE, ßA3/A1-crystallin fails to translocate to the lysosomes, perhaps because misfolding of the mutant protein masks sorting signals required for proper trafficking. The digestion of phagocytized outer segments requires a high level of lysosomal enzyme activity, and cathepsin D, the major enzyme responsible for proteolysis of the outer segments, is decreased in mutant RPE cells. Interestingly, our results also indicate a defect in the autophagy process in the Nuc1 RPE, which is probably also linked to impaired lysosomal function, because phagocytosis and autophagy might share common mechanisms in degradation of their targets. ßA3/A1-crystallin is a novel lysosomal protein in RPE, essential for degradation of phagocytosed material.
Asunto(s)
Cristalinas/genética , Mutación , Fagosomas/genética , Epitelio Pigmentado de la Retina/metabolismo , Animales , Cristalinas/metabolismo , Fagosomas/metabolismo , Fagosomas/ultraestructura , Ratas , Ratas Sprague-Dawley , Epitelio Pigmentado de la Retina/ultraestructuraRESUMEN
Purpose: Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly worldwide. Clinical imaging and histopathologic studies are crucial to understanding disease pathology. This study combined clinical observations of three brothers with geographic atrophy (GA), followed for 20 years, with histopathologic analysis. Methods: For two of the three brothers, clinical images were taken in 2016, 2 years prior to death. Immunohistochemistry, on both flat-mounts and cross sections, histology, and transmission electron microscopy were used to compare the choroid and retina in GA eyes to those of age-matched controls. Results: Ulex europaeus agglutinin (UEA) lectin staining of the choroid demonstrated a significant reduction in the percent vascular area and vessel diameter. In one donor, histopathologic analysis demonstrated two separate areas with choroidal neovascularization (CNV). Reevaluation of swept-source optical coherence tomography angiography (SS-OCTA) images revealed CNV in two of the brothers. UEA lectin also revealed a significant reduction in retinal vasculature in the atrophic area. A subretinal glial membrane, composed of processes positive for glial fibrillary acidic protein and/or vimentin, occupied areas identical to those of retinal pigment epithelium (RPE) and choroidal atrophy in all three AMD donors. SS-OCTA also demonstrated presumed calcific drusen in the two donors imaged in 2016. Immunohistochemical analysis and alizarin red S staining verified calcium within drusen, which was ensheathed by glial processes. Conclusions: This study demonstrates the importance of clinicohistopathologic correlation studies. It emphasizes the need to better understand how the symbiotic relationship between choriocapillaris and RPE, glial response, and calcified drusen impact GA progression.
Asunto(s)
Neovascularización Coroidal , Atrofia Geográfica , Degeneración Macular , Masculino , Anciano , Humanos , Atrofia Geográfica/diagnóstico , Hermanos , Retina/diagnóstico por imagen , Epitelio Pigmentado de la RetinaRESUMEN
In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options.Abbreviations: ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe2+: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; H2O2: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1ß: interleukin 1 beta; IS: Inner segment; ITGB1/integrin ß1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.
Asunto(s)
Ferroptosis , Degeneración Macular , Animales , Humanos , Ratones , Anticuerpos Monoclonales , Autofagia/fisiología , Inflamasomas/metabolismo , Lipocalina 2/genética , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Ratones Endogámicos NOD , Ratones SCID , Nucleotidiltransferasas/metabolismoRESUMEN
Retinal vascular development is a complex process that is not yet fully understood. The majority of research in this area has focused on astrocytes and the template they form in the inner retina, which precedes endothelial cells in the mouse retina. In humans and dogs, however, astrocyte migration follows behind development of blood vessels, suggesting that other cell types may guide this process. One such cell type is the ganglion cell, which differentiates before blood vessel formation and lies adjacent to the primary retinal vascular plexus. The present study investigated the potential role played by ganglion cells in vascular development using Math5(-/-) mice. It has previously been reported that Math5 regulates the differentiation of ganglion cells and Math5(-/-) mice have a 95% reduction in these cells. The development of blood vessels and glia was investigated using Griffonia simplicifolia isolectin B4 labeling and GFAP immunohistochemistry, respectively. JB-4 analysis demonstrated that the hyaloid vessels arose from choriovitreal vessels adjacent to the optic nerve area. As previously reported, Math5(-/-) mice had a rudimentary optic nerve. The primary retinal vessels did not develop post-natally in the Math5(-/-) mice, however, branches of the hyaloid vasculature eventually dove into the retina and formed the inner retinal capillary networks. An astrocyte template only formed in some areas of the Math5(-/-) retina. In addition, GFAP(+) Müller cells were seen throughout the retina that had long processes wrapped around the hyaloid vessels. Transmission electron microscopy confirmed Müller cell abnormalities and revealed disruptions in the inner limiting membrane. The present data demonstrates that the loss of ganglion cells in the Math5(-/-) mice is associated with a lack of retinal vascular development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuroglía/patología , Células Ganglionares de la Retina/fisiología , Vasos Retinianos/patología , Animales , Animales Recién Nacidos , Técnica del Anticuerpo Fluorescente Indirecta , Eliminación de Gen , Técnicas de Genotipaje , Proteína Ácida Fibrilar de la Glía , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía Electrónica de Transmisión , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/metabolismo , Vítreo Primario Hiperplásico Persistente/fisiopatología , Lectinas de Plantas/metabolismo , Neovascularización Retiniana/fisiopatología , Vasos Retinianos/metabolismoRESUMEN
Choroideremia (CHM) is a recessive, X-linked disease that affects 1 in 50,000 people worldwide. CHM causes night blindness in teenage years with vision loss progressing over the next two to three decades. While CHM is known to cause progressive loss of retinal pigment epithelial (RPE) cells, photoreceptors and choroidal vessels, little attention has been given to retinal glial changes in eyes with CHM. In addition, while choroidal loss has been observed clinically, no histopathologic assessment of choroidal loss has been done. We investigated glial remodeling and activation as well as choriocapillaris changes and their association with RPE loss in postmortem eyes from two donors with CHM. Eyes were fixed and cryopreserved or the retina and choroid/RPE were processed as flatmounts with a small piece cut for transmission electron microscopy. A dense glial membrane, made up of vimentin and GFAP double-positive cells, occupied the subretinal space in the area of RPE and photoreceptor loss of both eyes. The membranes did not extend into the far periphery, where RPE and photoreceptors were viable. A glial membrane was also found on the vitreoretinal surface. Transmission electron microscopy analysis demonstrated prominence and disorganization of glial cells, which contained exosome-like vesicles. UEA lectin demonstrated complete absence of choriocapillaris in areas with RPE loss while some large choroidal vessels remained viable. In the far periphery, where the RPE monolayer was intact, choriocapillaris appeared normal. The extensive glial remodeling present in eyes with CHM should be taken into account when therapies such as stem cell replacement are considered as it could impede cells entering the retina. This gliosis would also need to be reversed to some extent for Müller cells to perform their normal homeostatic functions in the retina. Future studies investigating donor eyes as well as clinical imaging from carriers or those with earlier stages of CHM will prove valuable in understanding the glial changes, which could affect disease progression if they occur early. This would also provide insights into the progression of disease in the photoreceptor/RPE/choriocapillaris complex, which is crucial for identifying new treatments and finding the windows for treatment.
RESUMEN
BACKGROUND: Valuable insights into the complex process of retinal vascular development can be gained using models with abnormal retinal vasculature. Two such models are the recently described mouse lines with mutations in Lama1, an important component of the retinal internal limiting membrane (ILM). These mutants have a persistence of the fetal vasculature of vitreous (FVV) but lack a primary retinal vascular plexus. The present study provides a detailed analysis of astrocyte and vascular development in these Lama1 mutants. RESULTS: Although astrocytes and blood vessels initially migrate into Lama1 mutant retinas, both traverse the peripapillary ILM into the vitreous by P3. Once in the vitreous, blood vessels anastomose with vessels of the vasa hyaloidea propria, part of the FVV, and eventually re-enter the retina where they dive to form the inner and outer retinal capillary networks. Astrocytes continue proliferating within the vitreous to form a dense mesh that resembles epiretinal membranes associated with persistent fetal vasculature and proliferative vitreoretinopathy. CONCLUSIONS: Lama1 and a fully intact ILM are required for normal retinal vascular development. Mutations in Lama1 allow developing retinal vessels to enter the vitreous where they anastomose with vessels of the hyaloid system which persist and expand. Together, these vessels branch into the retina to form fairly normal inner retinal vascular capillary plexi. The Lama1 mutants described in this report are potential models for studying the human conditions persistent fetal vasculature and proliferative vitreoretinopathy.
Asunto(s)
Membrana Epirretinal/metabolismo , Laminina/genética , Mutación , Vasos Retinianos/crecimiento & desarrollo , Cuerpo Vítreo/irrigación sanguínea , Animales , Membrana Epirretinal/embriología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Vasos Retinianos/embriología , Vitreorretinopatía Proliferativa/embriología , Vitreorretinopatía Proliferativa/genética , Vitreorretinopatía Proliferativa/metabolismo , Cuerpo Vítreo/embriología , Cuerpo Vítreo/crecimiento & desarrolloRESUMEN
The purpose of this study was to develop and characterize a rat model of choroidal neovascularization (CNV) as occurs in age-related macular degeneration. The lipid hydroperoxide 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (HpODE) is found in submacular Bruch's membrane in aged humans and has been reported to generate neovascularization in a rabbit model. Three weeks after a single subretinal injection of 30 microg of HpODE, eyes of Sprague-Dawley rats were harvested. Follow-up fluorescein angiography was done on other animals until 5 weeks postinjection. Histological studies, immunohistochemical staining, and flatmount choroids for CNV measurements were performed. In addition, we used murine neuronal, bovine endothelial, and human ARPE19 cells for testing the in vitro effects of HpODE. CNV developed in 85.7% of HpODE-injected eyes. The neovascular areas were significantly greater in HpODE-injected eyes compared with those in control eyes (P = 0.023). The CNV had maximum dye leakage at 3 weeks, which subsided by the 5th week. Histologically, CNV extended from the choriocapillaris into the subretinal space. ED1-positive macrophages were recruited to the site. In vitro assays demonstrated that only 30 ng/ml HpODE induced cell proliferation and migration of endothelial cells. HpODE-induced CNV was highly reproducible, and its natural course seems to be ideal for evaluating therapeutic modalities. Because HpODE has been isolated from aged humans, the HpODE-induced rat model seems to be a relevant experimental model for CNV in age-related macular degeneration.
Asunto(s)
Neovascularización Coroidal/inducido químicamente , Neovascularización Coroidal/patología , Modelos Animales de Enfermedad , Peróxidos Lipídicos/efectos adversos , Degeneración Macular/patología , Animales , Bovinos , Línea Celular , Proliferación Celular , Coroides/patología , Coroides/ultraestructura , Relación Dosis-Respuesta a Droga , Humanos , Inyecciones , Masculino , Ratones , Conejos , Ratas , Ratas Sprague-Dawley , Retina/patología , Retina/ultraestructuraRESUMEN
PURPOSE: This article describes the first retinal histopathologic findings in a patient with Susac's syndrome (SS). DESIGN: Observational case report. PARTICIPANT: A 51-year-old white woman diagnosed with SS. METHODS: Eyes from a 51-year-old white woman diagnosed with SS were obtained at autopsy. One retina was dissected and processed for adenosine diphosphatase (ADPase) flat embedding. Selected areas were processed further for transmission electron microscopy. MAIN OUTCOME MEASURES: Histopathologic examination using ADPase flat-embedding technique. RESULTS: There were vaso-occlusive changes in the retinal periphery resulting in small areas of capillary dropout. Cross-sections demonstrated serous filled spaces between the retinal blood vessels and the internal limiting membrane. Lumens adjacent to these spaces appeared compressed and sometimes closed, but without thrombosis. Decreased ADPase activity in some peripheral blood vessels suggested endothelial cell dysfunction and vaso-occlusion. In the optic nerve head, numerous corpora amylacea were observed in the vicinity of capillaries with thickened walls and narrow lumens. Transmission electron microscopy demonstrated thickened and amorphous vascular basal lamina and open endothelial cell junctions in some retinal blood vessels. CONCLUSIONS: The serous deposits with compression of retinal vessel lumens observed histologically probably represent the so-called string of pearls described clinically in SS. Chronic extension of these serous deposits along the vessel wall possibly are the cause of retinal arterial wall plaques as described by Gass and other investigators. In the optic nerve head, corpora amylacea are probably a result of microinfarcts resulting from optic nerve head capillary angiopathy. Accumulation of amorphous material in the basal lamina, loss of viable endothelial cells, and capillary dropout suggest that SS may be an endotheliopathy.
Asunto(s)
Disco Óptico/ultraestructura , Enfermedades del Nervio Óptico/diagnóstico , Enfermedades de la Retina/diagnóstico , Vasos Retinianos/ultraestructura , Síndrome de Susac/diagnóstico , Apirasa/metabolismo , Femenino , Humanos , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Disco Óptico/enzimología , Enfermedades del Nervio Óptico/enzimología , Enfermedades de la Retina/enzimología , Vasos Retinianos/enzimologíaRESUMEN
Purpose: This study evaluates whether topical ketotifen fumarate (KTF) can prevent geographic atrophy (GA)-like phenotypes in a rat model. Methods: Pharmacokinetics (PKs) of KTF after topical administration twice daily for 5 days was analyzed in rat retina, retinal pigment epithelium (RPE)/choroid/sclera, and in plasma by an liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Rats were then given hydrogel implants +/- 48/80 in the superior subconjunctival space and topically treated with 1% and 0.25% of KTF or phosphate buffer saline (PBS) twice daily. Rats were euthanized at 1, 2, 4, and 8 weeks postinjection. Choroidal mast cells (MCs) were stained with nonspecific esterase and the RPE monolayer was labeled with RPE65 and ZO-1 in whole mount choroids. Retinal and choroidal areas were determined in cryosections stained with picrosirius red. Dark-adapted electroretinogram (ERG) was also performed to evaluate retinal function. Results: PK results showed the highest level of KTF (average 5.6 nM/mg) in the RPE/choroid/sclera in rats given topical 1% KTF. Topical 1% KTF significantly reduced choroidal MC degranulation at 1 week and 2 weeks (both P < 0.001) and RPE loss at 4 weeks (P < 0.001) as well as retinal and choroidal thinning (both P < 0.001) and reduction in ERG amplitude at 8 weeks (P < 0.05) compared to PBS. Similar results were obtained with 0.25% KTF. Conclusions: Both 1% and 0.25% KTF eye drops effectively reduced MC degranulation, RPE loss, and retinal and choroidal thinning while preventing the decline of ERG amplitude in a GA-like rat model. These data suggest that topical KTF might be a new therapeutic drug for treating GA. Translational Relevance: The results of this study demonstrate that topical KTF successfully reduced GA-like phenotypes in a rat model and may provide a novel therapy for GA.
Asunto(s)
Atrofia Geográfica , Animales , Degranulación de la Célula , Coroides , Cromatografía Liquida , Células Epiteliales , Atrofia Geográfica/tratamiento farmacológico , Cetotifen/farmacología , Ratas , Pigmentos Retinianos , Espectrometría de Masas en TándemRESUMEN
The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for ßA3/A1-crystallin in RPE. ßA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that ßA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPß) and that ßA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that ßA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPß/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.
Asunto(s)
Polaridad Celular/fisiología , Endocitosis , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Cadena A de beta-Cristalina/metabolismo , Animales , Polaridad Celular/genética , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/ultraestructura , Humanos , Ratones Noqueados , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosforilación , Unión Proteica , Epitelio Pigmentado de la Retina/citología , Cadena A de beta-Cristalina/genéticaRESUMEN
Here, we report that the functionality of vascular progenitors (VP) generated from normal and disease-primed conventional human induced pluripotent stem cells (hiPSC) can be significantly improved by reversion to a tankyrase inhibitor-regulated human naïve epiblast-like pluripotent state. Naïve diabetic vascular progenitors (N-DVP) differentiated from patient-specific naïve diabetic hiPSC (N-DhiPSC) possessed higher vascular functionality, maintained greater genomic stability, harbored decreased lineage-primed gene expression, and were more efficient in migrating to and re-vascularizing the deep neural layers of the ischemic retina than isogenic diabetic vascular progenitors (DVP). These findings suggest that reprogramming to a stable naïve human pluripotent stem cell state may effectively erase dysfunctional epigenetic donor cell memory or disease-associated aberrations in patient-specific hiPSC. More broadly, tankyrase inhibitor-regulated naïve hiPSC (N-hiPSC) represent a class of human stem cells with high epigenetic plasticity, improved multi-lineage functionality, and potentially high impact for regenerative medicine.
Asunto(s)
Vasos Sanguíneos/patología , Diabetes Mellitus/patología , Células Madre Pluripotentes Inducidas/patología , Isquemia/terapia , Retina/patología , Células Madre/patología , Tanquirasas/antagonistas & inhibidores , Adulto , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Linaje de la Célula/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Daño del ADN , Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Código de Histonas , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Isquemia/patología , Ratones , Organoides/efectos de los fármacos , Organoides/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Regiones Promotoras Genéticas/genética , Células Madre/efectos de los fármacos , Células Madre/ultraestructura , Tanquirasas/metabolismo , Teratoma/patología , Transcripción Genética/efectos de los fármacosRESUMEN
Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress normally. The regulatory mechanisms responsible for fetal vascular regression remain obscure, as does the underlying cause of regression failure. However, there are a few animal models that mimic the clinical manifestations of human PFV, which can be used to study different aspects of the disease. One such model is the Nuc1 rat model that arose from a spontaneous mutation in the Cryba1 (crystallin, beta 1) gene and exhibits complete failure of the hyaloid vasculature to regress. Our studies with the Nuc1 rat indicate that macroautophagy/autophagy, a process in eukaryotic cells for degrading dysfunctional components to ensure cellular homeostasis, is severely impaired in Nuc1 ocular astrocytes. Further, we show that CRYBA1 interacts with EGFR (epidermal growth factor receptor) and that loss of this interaction in Nuc1 astrocytes increases EGFR levels. Moreover, our data also show a reduction in EGFR degradation in Nuc1 astrocytes compared to control cells that leads to over-activation of the mechanistic target of rapamycin kinase complex 1 (MTORC1) pathway. The impaired EGFR-MTORC1-autophagy signaling in Nuc1 astrocytes triggers abnormal proliferation and migration. The abnormally migrating astrocytes ensheath the hyaloid artery, contributing to the pathogenesis of PFV in Nuc1, by adversely affecting the vascular remodeling processes essential to regression of the fetal vasculature. Herein, we demonstrate in vivo that gefitinib (EGFR inhibitor) can rescue the PFV phenotype in Nuc1 and may serve as a novel therapy for PFV disease by modulating the EGFR-MTORC1-autophagy pathway. ABBREVIATIONS: ACTB: actin, beta; CCND3: cyclin 3; CDK6: cyclin-dependent kinase 6; CHQ: chloroquine; COL4A1: collagen, type IV, alpha 1; CRYBA1: crystallin, beta A1; DAPI: 4'6-diamino-2-phenylindole; EGFR: epidermal growth factor receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; KDR: kinase insert domain protein receptor; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MKI67: antigen identified by monoclonal antibody Ki 67; MTORC1: mechanistic target of rapamycin kinase complex 1; PARP: poly (ADP-ribose) polymerase family; PCNA: proliferating cell nuclear antigen; PFV: persistent fetal vasculature; PHPV: persistent hyperplastic primary vitreous; RPE: retinal pigmented epithelium; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SQSTM1/p62: sequestome 1; TUBB: tubulin, beta; VCL: vinculin; VEGFA: vascular endothelial growth factor A; WT: wild type.
Asunto(s)
Astrocitos/metabolismo , Autofagia/genética , Receptores ErbB/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Vítreo Primario Hiperplásico Persistente/metabolismo , Cadena A de beta-Cristalina/metabolismo , Animales , Astrocitos/efectos de los fármacos , Autofagia/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Receptores ErbB/antagonistas & inhibidores , Ojo/metabolismo , Gefitinib/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/genética , Lisosomas/metabolismo , Lisosomas/ultraestructura , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Microscopía Inmunoelectrónica , Morfolinas/farmacología , Vítreo Primario Hiperplásico Persistente/genética , Vítreo Primario Hiperplásico Persistente/patología , Vítreo Primario Hiperplásico Persistente/terapia , Ratas , Transducción de Señal/genética , Sirolimus/farmacología , Cadena A de beta-Cristalina/genéticaRESUMEN
BACKGROUND: Acanthamoeba scleritis is an uncommon but severe complication of acanthamoeba keratitis. We report the clinical and histopathologic features of a patient with acanthamoeba sclerokeratitis. METHODS: Review of the patient's clinical records and histopathologic examination of the globe including light microscopy and transmission electron microscopy. RESULTS: Review of the clinical record of the patient revealed a past ocular history of penetrating keratoplasty for persistent acanthamoeba keratitis. Later in the course of treatment, the patient developed nodular necrotizing scleritis with culture-proven viable acanthamoeba in this area. She underwent enucleation of the eye for persistent acanthamoeba sclerokeratitis. Histopathologic examination of the globe revealed no acanthamoeba cysts or trophozoites at the site of crotherapy. CONCLUSION: Our study provides evidence for the invasion of acanthamoeba organisms into the sclera in a case of acanthamoeba keratitis. The presence of trophozites in scleral tissue may exacerbate the immune response leading to nodular scleritis.
Asunto(s)
Queratitis por Acanthamoeba/complicaciones , Queratitis por Acanthamoeba/patología , Acanthamoeba , Escleritis/patología , Escleritis/parasitología , Acanthamoeba/crecimiento & desarrollo , Acanthamoeba/ultraestructura , Queratitis por Acanthamoeba/tratamiento farmacológico , Adulto , Animales , Antiinflamatorios/uso terapéutico , Antiparasitarios/uso terapéutico , Lentes de Contacto Hidrofílicos , Crioterapia , Femenino , Humanos , Microscopía Electrónica de Transmisión , Esclerótica/patología , Esclerótica/ultraestructura , Escleritis/terapia , Trofozoítos/ultraestructuraRESUMEN
PURPOSE: Prior investigation has demonstrated that angioblasts are present in the inner retinas of human embryos and fetuses and that they differentiate and organize to form the primordial retinal vasculature. The purpose of this study was to characterize these angioblasts further and examine ligands that might control their migration and differentiation. METHODS: Immunohistochemistry was used to localize stroma-derived factor-1 (SDF-1), its receptor CXCR4, stem cell factor (SCF), and its receptor c-Kit on sections obtained from human eyes at from 6 to 23 weeks' gestation (WG). Coexpression of CD39 (marker for retinal angioblasts and endothelial cells) and CXCR4 or c-Kit was investigated by confocal microscopy. RESULTS: SDF-1 was prominent in inner retina with the greatest reaction product near the internal limiting membrane (ILM). SCF immunoreactivity was also confined to the inner retina and increased significantly between 7 and 12 WG. The level of both ligands declined by 22 WG. A layer of CXCR4(+) and c-Kit(+) precursors, some of which coexpressed CD39, existed in the inner retina from 7 to 12 WG. With migration, c-Kit was downregulated, whereas CD39(+) cells continued to express CXCR4 as they formed cords. With canalization, CXCR4 expression was downregulated. CONCLUSIONS: Embryonic human retina has a pool of precursors (CXCR4(+) and c-Kit(+)) that enlarged centrifugally during fetal development. From this pool emerges angioblasts, which migrate anteriorly into the nerve fiber layer where SDF-1 and SCF levels are highest. c-Kit expression declines with apparent migration, and CXCR4 expression declines with canalization of new vessels. Both SCF and SDF-1 are associated with the differentiation of retinal precursors into angioblasts and their migration to sites of vessel assembly.