To investigate the major organelles of the retinal pigment epithelium (RPE) in wild-type (WT, control) mice and their changes in pigmented Abca4 knockout (Abca4-/-) mice with in situ morphologic, ...spatial, and spectral characterization of live ex vivo flat-mounted RPE using multicolor confocal fluorescence microscopy (MCFM).
In situ imaging of RPE flat-mounts of agouti Abca4-/- (129S4), agouti WT (129S1/SvlmJ) controls, and B6 albino mice (C57BL/6J-Tyrc-Brd) was performed with a Nikon A1 confocal microscope. High-resolution confocal image z-stacks of the RPE cell mosaic were acquired with four different excitation wavelengths (405 nm, 488 nm, 561 nm, and 640 nm). The autofluorescence images of RPE, including voxel-by-voxel emission spectra, were acquired and processed with Nikon NIS-AR Elements software.
The 3-dimensional multicolor confocal images provided a detailed visualization of the RPE cell mosaic, including its melanosomes and lipofuscin granules, and their varying characteristics in the different mice strains. The autofluorescence spectra, spatial distribution, and morphologic features of melanosomes and lipofuscin granules were measured. Increased numbers of lipofuscin and reduced numbers of melanosomes were observed in the RPE of Abca4-/- mice relative to controls.
A detailed assessment of the RPE autofluorescent granules and their changes ex vivo was possible with MCFM. For all excitation wavelengths, autofluorescence from the RPE cells was predominantly contributed by lipofuscin granules, while melanosomes were found to be essentially nonfluorescent. The red shift of the emission peak confirmed the presence of multiple chromophores within lipofuscin granules. The elevated autofluorescence levels in Abca4-/- mice correlated well with the increased number of lipofuscin granules.
Mitochondrial disorders preferentially affect tissues with high energy requirements, such as the retina and corneal endothelium, in human eyes. Mesenchymal stem cell (MSC)-based treatment has been ...demonstrated to be beneficial for ocular degeneration. However, aside from neuroprotective paracrine actions, the mechanisms underlying the beneficial effect of MSCs on retinal and corneal tissues are largely unknown. In this study, we investigated the fate and associated characteristics of mitochondria subjected to intercellular transfer from MSCs to ocular cells.
MSCs were cocultured with corneal endothelial cells (CECs), 661W cells (a photoreceptor cell line) and ARPE-19 cells (a retinal pigment epithelium cell line). Immunofluorescence, fluorescence activated cell sorting and confocal microscopy imaging were employed to investigate the traits of intercellular mitochondrial transfer and the fate of transferred mitochondria. The oxygen consumption rate of recipient cells was measured to investigate the effect of intercellular mitochondrial transfer. Transcriptome analysis was performed to investigate the expression of metabolic genes in recipient cells with donated mitochondria.
Mitochondrial transport is a ubiquitous intercellular mechanism between MSCs and various ocular cells, including the corneal endothelium, retinal pigmented epithelium, and photoreceptors. Additionally, our results indicate that the donation process depends on F-actin-based tunneling nanotubes. Rotenone-pretreated cells that received mitochondria from MSCs displayed increased aerobic capacity and upregulation of mitochondrial genes. Furthermore, living imaging determined the ultimate fate of transferred mitochondria through either degradation by lysosomes or exocytosis as extracellular vesicles.
For the first time, we determined the characteristics and fate of mitochondria undergoing intercellular transfer from MSCs to various ocular cells through F-actin-based tunneling nanotubes, helping to characterize MSC-based treatment for ocular tissue regeneration.
Retinal pigment epithelium (RPE) cells show heterogeneous levels of pigmentation when cultured in vitro. To know whether their color in appearance is correlated with the function of the RPE, we ...analyzed the color intensities of human-induced pluripotent stem cell-derived RPE cells (iPSC-RPE) together with the gene expression profile at the single-cell level. For this purpose, we utilized our recent invention, Automated Live imaging and cell Picking System (ALPS), which enabled photographing each cell before RNA-sequencing analysis to profile the gene expression of each cell. While our iPSC-RPE were categorized into four clusters by gene expression, the color intensity of iPSC-RPE did not project any specific gene expression profiles. We reasoned this by less correlation between the actual color and the gene expressions that directly define the level of pigmentation, from which we hypothesized the color of RPE cells may be a temporal condition not strongly indicating the functional characteristics of the RPE.
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•A new series of Bi4-xCexTi3O12 reddish brown to dark yellow NIR reflecting pigments with UV shielding properties have been synthesised.•The doping of Ce ions tunes the colour of the ...bismuth titanate from ivory to reddish brown.•The Bi3.75Ce0.25Ti3O12 reddish brown pigment shows IR reflectance about 71.79%.•All the synthesised pigments showed equally proficient UV absorbance value of 1.271 at 274 nm.•The Bi3.75Ce0.25Ti3O12 reddish brown pigment was used in thermosetting acrylic resin paint and the ability of its thermal resistance was analysed.
A new series of Bi4-xCexTi3O12 (0.25, 0.50, 0.75, 1.0, 1.25) reddish brown to dark yellow NIR reflectance pigments with UV shielding properties have been synthesised via citrate auto combustion synthesis. The doping of Ce ions tunes the colour of the bismuth titanate from ivory to reddish brown. The reddish brown Bi3.75Ce0.25Ti3O12 pigment shows NIR reflectance of around 71.79%. All the synthesized pigments shows equally proficient UV absorbance value 1.271 at 274 nm. The increasing Ce ion concentration changes the colour from darker to lighter reddish shades. This reddish brown pigment was used in thermosetting acrylic resin paint and the ability of its heat resistance was analysed. The higher thermal stability and desirable optical properties of the synthesised pigments enables them as cost effective, environmental friendly NIR reflective reddish brown cool pigments and good alternative to the toxic pigments of the same colour.
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.
Transplantation of autologous human induced pluripotent stem cell-derived retinal pigment epithelial (hiPSC-RPE) sheets is a promising therapy for age-related macular degeneration (AMD). As melanin ...content is a representative feature of healthy RPE, we used polarization-sensitive optical coherence tomography (PS-OCT) to estimate the relative melanin content of RPE in diseased and non-diseased area, and in human iPSC-RPE sheets in vitro and in vivo by evaluating the randomness of polarization (entropy). Two aged Japanese women, one with neovascular AMD that underwent transplantation of an autologous hiPSC-RPE cell sheet and another with binocular dry AMD, were selected for this study. Entropy value was minimal in cells containing no melanin, whereas that of human RPE and hiPSC-RPE sheets was high. En face entropy of the cultured hiPSC-RPE sheet was compared with its grey-scale photo and its values were found to be inversely correlated with the extent of absence of pigmentation in vitro. En face entropy maps were compared to colour fundus photographs, fundus autofluorescence images, and fluorescein angiography images from patients. Entropy values of intact and defective RPEs and of iPSC-RPE transplant areas were determined in vivo using PS-OCT B-scan images. PS-OCT was found to be applicable in the estimation of relative melanin content of cultured and transplanted RPEs in regenerative medicine.
Age-related macular degeneration (AMD) is the primary cause of blindness in adults over 60 years of age, and clinical trials are currently assessing the therapeutic potential of retinal pigmented ...epithelial (RPE) cell monolayers on implantable scaffolds to treat this disease. However, challenges related to the culture, long-term storage, and long-distance transport of such implants currently limit the widespread use of adherent RPE cells as therapeutics. Here we report a xeno-free protocol to cryopreserve a confluent monolayer of clinical-grade, human embryonic stem cell-derived RPE cells on a parylene scaffold (REPS) that yields viable, polarized, and functional RPE cells post-thaw. Thawed cells exhibit ≥ 95% viability, have morphology, pigmentation, and gene expression characteristic of mature RPE cells, and secrete the neuroprotective protein, pigment epithelium-derived factor (PEDF). Stability under liquid nitrogen (LN
) storage has been confirmed through one year. REPS were administered immediately post-thaw into the subretinal space of a mammalian model, the Royal College of Surgeons (RCS)/nude rat. Implanted REPS were assessed at 30, 60, and 90 days post-implantation, and thawed cells demonstrate survival as an intact monolayer on the parylene scaffold. Furthermore, immunoreactivity for the maturation marker, RPE65, significantly increased over the post-implantation period in vivo, and cells demonstrated functional attributes similar to non-cryopreserved controls. The capacity to cryopreserve adherent cellular therapeutics permits extended storage and stable transport to surgical sites, enabling broad distribution for the treatment of prevalent diseases such as AMD.
IMPORTANCE: Recent studies have linked a vision-threatening maculopathy with long-term use of pentosan polysulfate sodium (PPS). OBJECTIVE: To evaluate the disease course in PPS-associated ...maculopathy after drug cessation. DESIGN, SETTING, AND PARTICIPANTS: In this retrospective case series, patients diagnosed with PPS-associated maculopathy with at least 6 months of follow-up after drug cessation who were treated at the Emory Eye Center, Atlanta, Georgia, or the Casey Eye Institute, Portland, Oregon, were included. Data were collected from April 2014 through November 2019. MAIN OUTCOMES AND MEASURES: Change in visual acuity and retinal imaging characteristics over time. RESULTS: Of the 11 included patients, all were female, and the median (interquartile range IQR) age was 53 (44-63) years. Participants had a baseline visit at a median (IQR) of 2 (0-4) months after drug cessation and were subsequently observed for a median (IQR) of 12 (8-26) months. The median (IQR) cumulative PPS exposure was 1.97 (1.55-2.18) kg. No eyes exhibited a demonstrable improvement in disease after discontinuing PPS. A total of 9 of 11 patients (82%) reported worsening visual symptoms at the final visit. The mean (SD) logMAR visual acuity was 0.14 (0.23) and 0.14 (0.34) at the baseline and final visit, respectively. Visual acuity improved by 2 or more Snellen lines in 1 eye (5%) and declined by 2 or more Snellen lines in 2 eyes of 1 patient (9%). There was evolution in the pattern of fundus autofluorescence changes and/or optical coherence tomography findings in all eyes. A total of 17 eyes (77%) exhibited expansion of the area of involved tissue. A total of 7 eyes (32%) had macular retinal pigment epithelium atrophy at the baseline visit, and atrophy enlarged after discontinuation of PPS in all 7 eyes, with a median (IQR) growth rate of 0.32 (0.13-0.38) mm per year. CONCLUSIONS AND RELEVANCE: These retrospective data among 11 patients suggest PPS-associated maculopathy continues to evolve after drug cessation for at least 10 years. In some cases, progressive retinal pigment epithelium atrophy encroaches on the foveal center and thus may pose a long-term threat to central vision.
Age-related macular degeneration (AMD) is a devastating neurodegenerative disease and a major cause of blindness in the developed world. Owing to its complexity and the lack of an adequate human ...model that recapitulates key aspects of the disease, the molecular mechanisms of AMD pathogenesis remain poorly understood. Here we show that cultured human retinal pigment epithelium (RPE) from AMD donors (AMD RPE) are functionally impaired and exhibit distinct phenotypes compared with RPE cultured from normal donors (normal RPE). Accumulation of lipid droplets and glycogen granules, disintegration of mitochondria, and an increase in autophagosomes were observed in AMD RPE cultures. Compared with normal RPE, AMD RPE exhibit increased susceptibility to oxidative stress, produce higher levels of reactive oxygen species (ROS) under stress conditions, and showed reduced mitochondrial activity. Measurement of the ratio of LC3-II/ LC3-I, revealed impaired autophagy in AMD RPE as compared with normal RPE. Autophagic flux was also reduced in AMD RPE as compared with normal RPE, as shown by inability of AMD RPE to downregulate p62 levels during starvation. Impaired autophagic pathways were further shown by analyzing late autophagic vesicles; immunostaining with lysosome-associated membrane protein 1 (LAMP-1) antibody revealed enlarged and annular LAMP-1-positive organelles in AMD RPE as opposed to smaller discrete puncta observed in normal RPE. Our study provides insights into AMD cellular and molecular mechanisms, proposes dysfunctional autophagy as an underlying mechanism contributing to the pathophysiology of the disease, and opens up new avenues for development of novel treatment strategies.
This short review paper describes spectroscopic studies on pigment–pigment and pigment–protein interactions of chlorophyll (Chl)
a and
b bound to the recombinant protein of class IIa water soluble ...chlorophyll protein (WSCP) from cauliflower. Two Chls form a strongly excitonically coupled open sandwich dimer within the tetrameric protein matrix. In marked contrast to the mode of excitonic coupling of Chl and bacterio-Chl molecules in light harvesting complexes and reaction centers of all photosynthetic organisms, the unique structural pigment array in the Chl dimer of WSCP gives rise to an upper excitonic state with a large oscillator strength. This property opens the way for thorough investigations on exciton relaxation processes in Chl–protein complexes.
Lifetime measurements of excited singlet states show that the unusual stability towards photodamage of Chls bound to WSCP, which lack any protective carotenoid molecule, originates from a high diffusion barrier to interaction of molecular dioxygen with Chl triplets.
Site selective spectroscopic methods provide a wealth of information on the interactions of the Chls with the protein matrix and on the vibronic structure of the pigments.
The presented data and discussions illustrate the great potential of WSCP as a model system for systematic experimental and theoretical studies on the functionalizing of Chls by the protein matrix. It opens the way for further detailed analyses and a deeper understanding of the properties of pigment protein complexes.
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