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Journal of Cell Science Sep 2020Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer...
Human retinal pigment epithelial-1 (RPE-1) cells are increasingly being used as a model to study mitosis because they represent a non-transformed alternative to cancer cell lines, such as HeLa cervical adenocarcinoma cells. However, the lack of an efficient method to synchronize RPE-1 cells in mitosis precludes their application for large-scale biochemical and proteomics assays. Here, we report a protocol to synchronize RPE-1 cells based on sequential treatments with the Cdk4 and Cdk6 inhibitor PD 0332991 (palbociclib) and the microtubule-depolymerizing drug nocodazole. With this method, the vast majority (80-90%) of RPE-1 cells arrested at prometaphase and exited mitosis synchronously after release from nocodazole. Moreover, the cells fully recovered and re-entered the cell cycle after the palbociclib-nocodazole block. Finally, we show that this protocol could be successfully employed for the characterization of the protein-protein interaction network of the kinetochore protein Ndc80 by immunoprecipitation coupled with mass spectrometry. This synchronization method significantly expands the versatility and applicability of RPE-1 cells to the study of cell division and might be applied to other cell lines that do not respond to treatments with DNA synthesis inhibitors.
Topics: Humans; Kinetochores; Mitosis; Nocodazole; Prometaphase; Retinal Pigments
PubMed: 32878943
DOI: 10.1242/jcs.247940 -
Autophagy Feb 2022Diabetic retinopathy (DR) is a serious complication of diabetes mellitus and currently one of the major causes of blindness. Several previous studies have demonstrated...
Diabetic retinopathy (DR) is a serious complication of diabetes mellitus and currently one of the major causes of blindness. Several previous studies have demonstrated that autophagy, which is regulated by HMGB1 (high mobility group box 1), is involved in DR development. However, the role of autophagy in DR is quite complicated in that it promotes pericyte survival in early DR, whereas excessive autophagy causes excess stress and leads to necrosis. Therefore, this study aimed to investigate the relationship between HMGB1, the macroautophagy/autophagy-lysosome pathway, and DR, as well as their underlying molecular mechanisms. In brief, the relationship between high glucose (HG) and the autophagy-lysosome pathway was examined in retinal pigment epithelial (RPE) cells. The relationship was studied by detecting classical autophagic features, and siRNAs targeting HMGB1 and pharmacological regulators were used to explore the role of the autophagy-lysosome pathway in DR development. The results demonstrated that HG inhibited autophagy and diminished the degradative capacity of autophagy due to lysosome membrane permeabilization (LMP). In addition, HMGB1 was found to be involved in LMP via the CTSB (cathepsin B)-dependent pathway, but not the CTSL (cathepsin L)-dependent pathway. Knockdown of HMGB1 expression rescued LMP, restored the degradative capacity of autophagy, decreased the expression of inflammatory factors and VEGF (vascular endothelial growth factor), and protected against apoptosis in RPE cells in the early stages of DR.
Topics: Autophagy; Diabetes Mellitus; Diabetic Retinopathy; Down-Regulation; Epithelial Cells; HMGB1 Protein; Humans; Lysosomes; Retinal Pigments; Vascular Endothelial Growth Factor A
PubMed: 34024230
DOI: 10.1080/15548627.2021.1926655 -
Bioengineered Apr 2022Astragaloside-IV (AS-IV) (CHO) is a high-purity natural product extracted from , which has demonstrated biological activities. However, the effect of AS-IV on retinal...
Astragaloside-IV (AS-IV) (CHO) is a high-purity natural product extracted from , which has demonstrated biological activities. However, the effect of AS-IV on retinal pigment epithelial (RPE) cells in diabetic retinopathy (DR) remains unclear. In this study, high glucose (HG) was shown to promote ARPE-19 RPE cell death, increase the contents of reactive oxygen species (ROS) and oxidized glutathione (GSSG), and enhance lipid peroxidation density of mitochondrial membrane. In contrast, AS-IV decreased glutathione (GSH) content, mitochondria size and ridge. Addition of iron death inhibitor Ferrostatin-1 (Fer-1) to RPE cells decreased cell dead rate, thus indicating that HG-induced mitochondrial damage occurred due to ferroptosis. AS-IV alleviated HG-induced RPE cell damage. Furthermore, HG decreased levels of silent information regulator 1 (Sirt1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the nucleus of RPE cells; AS-IV could alleviate these effects and increased expression of glutathione peroxidase 4 (GPX4), glutamate cysteine ligase (GCLM) and glutamate cysteine ligase catalytic subunit (GCLC), which are Nrf2 downstream genes. Mechanistically, AS-IV was shown to alleviate the effects of HG by increasing mir-138-5p expression in RPE cells and promoting expression of Sirt1 and Nrf2 in the nucleus. Transfection of mir-138-5p agonist inhibited the regulatory effects of AS-IV on Sirt1 and Nrf2, accompanied by decreased GPX4, GCLM and GCLC levels, and restoration of ferroptosis-related changes. Collectively, HG increased ferroptosis rate in RPE cells. In addition, AS-IV inhibited miR-138-5p expression, subsequently increasing Sirt1/Nrf2 activity and cellular antioxidant capacity to alleviate ferroptosis, resulting decreased cell death, which potentially inhibits the DR pathological process.
Topics: Epithelial Cells; Ferroptosis; Glucose; Glutamate-Cysteine Ligase; MicroRNAs; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; Retinal Pigment Epithelium; Retinal Pigments; Sirtuin 1
PubMed: 35302431
DOI: 10.1080/21655979.2022.2049471 -
Cell Proliferation Apr 2022'Human retinal pigment epithelial cells' is the first set of guidelines on human retinal pigment epithelial cells in China, jointly drafted and agreed upon by experts...
'Human retinal pigment epithelial cells' is the first set of guidelines on human retinal pigment epithelial cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements and waste disposal requirements for human retinal pigment epithelial cells, which is applicable to quality control during the process of manufacturing and testing of human retinal pigment epithelial cells. It was originally released by the Chinese Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of human retinal pigment epithelial cells for applications.
Topics: China; Epithelial Cells; Humans; Neurons; Retinal Pigments
PubMed: 34773310
DOI: 10.1111/cpr.13153 -
Journal of Neuroinflammation Jul 2022We previously reported higher plasma levels of complement fragments C3a and C5a in neovascular Age-related Macular Degeneration (nAMD) patients with macular fibrosis....
BACKGROUND
We previously reported higher plasma levels of complement fragments C3a and C5a in neovascular Age-related Macular Degeneration (nAMD) patients with macular fibrosis. This study aimed to understand whether complement activation contributes to the development of macular fibrosis and the underlying mechanisms involved.
METHODS
Complement activation was blocked using a C5 neutralizing antibody (BB5.1) in C57BL/6J mice after induction of subretinal fibrosis using the two-stage laser protocol. Fibrotic lesions were examined 10 days after the 2nd laser through fundus examination and immunohistochemistry. The expression of C5aR in fibrotic lesions and retinal pigment epithelial (RPE) cultures were examined by confocal microscopy. Primary murine RPE cells were treated with C3a or C5a (10-100 ng/mL) or TGF-β2 (10 ng/mL). Epithelial-to-mesenchymal transition (EMT) was assessed through various readouts. The expression of E-cadherin, vimentin, fibronectin, α-SMA, Slug, ERK/AKT and pSMAD2/3 were determined by Western blot and immunocytochemistry. Collagen contraction and wound-healing assays were used as functional readouts of EMT. The production of IL-6, TGF-β1, TGF-β2 and VEGF by RPE cells were determined by ELISA. PMX53 was used to block C5aR in RPE cultures and in vivo in mice with subretinal fibrosis.
RESULTS
Extensive C5b-9 deposition was detected at the site of subretinal fibrosis. BB5.1 treatment completely abrogated complement activation and significantly reduced subretinal fibrosis. C5aR was detected in RPE and infiltrating MHC-II cells in subretinal fibrosis. In vitro, RPE cells constitutively express C5/C5a and C5aR, and their expression was increased by TGF-β2 treatment. C5a but not C3a increased fibronectin, α-SMA, vimentin and Slug expression, and decreased E-cadherin expression in RPE cells. C5a treatment also increased the contractility and migration of RPE cells and enhanced the production of VEGF and TGF-β1/2. C5a treatment induced pSmad2/3 and pERK1/2 expression in RPE cells and this was blocked by PMX53. PMX53 treatment significantly reduced sodium fluorescein leakage in the subretinal fibrosis model, while collagen-I lesions only mildly reduced.
CONCLUSIONS
Complement activation is critically involved in the development of subretinal fibrosis, partially through C5a-C5aR-mediated EMT in RPE cells. Targeting complement activation rather than C5a may be a novel approach for the management of macular fibrosis.
Topics: Animals; Cadherins; Collagen; Complement Activation; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibronectins; Fibrosis; Mice; Mice, Inbred C57BL; Retinal Pigment Epithelium; Retinal Pigments; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Vascular Endothelial Growth Factor A; Vimentin
PubMed: 35831910
DOI: 10.1186/s12974-022-02546-3 -
The Journal of Experimental Medicine Dec 2023Transplantation of retinal pigment epithelial (RPE) cells holds great promise for patients with retinal degenerative diseases, such as age-related macular degeneration....
Transplantation of retinal pigment epithelial (RPE) cells holds great promise for patients with retinal degenerative diseases, such as age-related macular degeneration. In-depth characterization of RPE cell product identity and critical quality attributes are needed to enhance efficacy and safety of replacement therapy strategies. Here, we characterized an adult RPE stem cell-derived (RPESC-RPE) cell product using bulk and single-cell RNA sequencing (scRNA-seq), assessing functional cell integration in vitro into a mature RPE monolayer and in vivo efficacy by vision rescue in the Royal College of Surgeons rats. scRNA-seq revealed several distinct subpopulations in the RPESC-RPE product, some with progenitor markers. We identified RPE clusters expressing genes associated with in vivo efficacy and increased cell integration capability. Gene expression analysis revealed lncRNA (TREX) as a predictive marker of in vivo efficacy. TREX knockdown decreased cell integration while overexpression increased integration in vitro and improved vision rescue in the RCS rats.
Topics: Animals; Rats; Biomarkers; Gene Expression Profiling; Neurons; Epithelial Cells; Retinal Pigments
PubMed: 37728563
DOI: 10.1084/jem.20230913 -
Biomolecules May 2023Age-related macular degeneration (AMD) causes vision loss in the elderly population. Dry AMD leads to the formation of Drusen, while wet AMD is characterized by cell... (Review)
Review
Age-related macular degeneration (AMD) causes vision loss in the elderly population. Dry AMD leads to the formation of Drusen, while wet AMD is characterized by cell proliferation and choroidal angiogenesis. The retinal pigment epithelium (RPE) plays a key role in AMD pathogenesis. In particular, helioreceptor renewal depends on outer segment phagocytosis of RPE cells, while RPE autophagy can protect cells from oxidative stress damage. However, when the oxidative stress burden is too high and homeostasis is disturbed, the phagocytosis and autophagy functions of RPE become damaged, leading to AMD development and progression. Hence, characterizing the roles of RPE cell phagocytosis and autophagy in the pathogenesis of AMD can inform the development of potential therapeutic targets to prevent irreversible RPE and photoreceptor cell death, thus protecting against AMD.
Topics: Aged; Humans; Phagocytosis; Autophagy; Macular Degeneration; Oxidative Stress; Epithelial Cells; Retinal Pigments
PubMed: 37371481
DOI: 10.3390/biom13060901 -
Trends in Neurosciences Jan 2022MicroRNAs are short, evolutionarily conserved noncoding RNAs that are critical for the control of normal cellular physiology. In the retina, photoreceptors are highly... (Review)
Review
MicroRNAs are short, evolutionarily conserved noncoding RNAs that are critical for the control of normal cellular physiology. In the retina, photoreceptors are highly specialized neurons that transduce light into electrical signals. Photoreceptors, however, are unable to process visual stimuli without the support of the retinal pigment epithelium (RPE). The RPE performs numerous functions to aid the retina, including the generation of visual chromophore and metabolic support. Recent work has underscored how microRNAs enable vision through their contributions to RPE functions. This review focuses on the biogenesis and control of microRNAs in rodents and humans, the roles microRNAs play in RPE function and degeneration, and how microRNAs could serve as potential therapeutics and biomarkers for visual diseases.
Topics: Humans; MicroRNAs; Retina; Retinal Pigment Epithelium; Retinal Pigments; Vision, Ocular
PubMed: 34753606
DOI: 10.1016/j.tins.2021.10.008 -
Progress in Retinal and Eye Research Mar 2011Cone photoreceptors mediate our daytime vision and function under bright and rapidly-changing light conditions. As their visual pigment is destroyed in the process of... (Review)
Review
Cone photoreceptors mediate our daytime vision and function under bright and rapidly-changing light conditions. As their visual pigment is destroyed in the process of photoactivation, the continuous function of cones imposes the need for rapid recycling of their chromophore and regeneration of their pigment. The canonical retinoid visual cycle through the retinal pigment epithelium cells recycles chromophore and supplies it to both rods and cones. However, shortcomings of this pathway, including its slow rate and competition with rods for chromophore, have led to the suggestion that cones might use a separate mechanism for recycling of chromophore. In the past four decades biochemical studies have identified enzymatic activities consistent with recycling chromophore in the retinas of cone-dominant animals, such as chicken and ground squirrel. These studies have led to the hypothesis of a cone-specific retina visual cycle. The physiological relevance of these studies was controversial for a long time and evidence for the function of this visual cycle emerged only in very recent studies and will be the focus of this review. The retina visual cycle supplies chromophore and promotes pigment regeneration only in cones but not in rods. This pathway is independent of the pigment epithelium and instead involves the Müller cells in the retina, where chromophore is recycled and supplied selectively to cones. The rapid supply of chromophore through the retina visual cycle is critical for extending the dynamic range of cones to bright light and for their rapid dark adaptation following exposure to light. The importance of the retina visual cycle is emphasized also by its preservation through evolution as its function has now been demonstrated in species ranging from salamander to zebrafish, mouse, primate, and human.
Topics: Animals; Humans; Light; Mice; Models, Biological; Primates; Retina; Retinal Cone Photoreceptor Cells; Retinal Pigment Epithelium; Retinal Pigments; Retinal Rod Photoreceptor Cells; Urodela; Vision, Ocular
PubMed: 21111842
DOI: 10.1016/j.preteyeres.2010.11.001 -
Cell Death & Disease Sep 2022Age-related macular degeneration (AMD) is a major vision-threatening disease. Although mesenchymal stem cells (MSCs) exhibit beneficial neural protective effects, their...
Age-related macular degeneration (AMD) is a major vision-threatening disease. Although mesenchymal stem cells (MSCs) exhibit beneficial neural protective effects, their limited differentiation capacity in vivo attenuates their therapeutic function. Therefore, the differentiation of MSCs into retinal pigment epithelial (RPE) cells in vitro and their subsequent transplantation into the subretinal space is expected to improve the outcome of cell therapy. Here, we transdifferentiated human umbilical cord MSCs (hUCMSCs) into induced RPE (iRPE) cells using a cocktail of five transcription factors (TFs): CRX, NR2E1, C-MYC, LHX2, and SIX6. iRPE cells exhibited RPE specific properties, including phagocytic ability, epithelial polarity, and gene expression profile. In addition, high expression of PTPN13 in iRPE cells endows them with an epithelial-to-mesenchymal transition (EMT)-resistant capacity through dephosphorylating syntenin1, and subsequently promoting the internalization and degradation of transforming growth factor-β receptors. After grafting into the subretinal space of the sodium iodate-induced rat AMD model, iRPE cells demonstrated a better therapeutic function than hUCMSCs. These results suggest that hUCMSC-derived iRPE cells may be promising candidates to reverse AMD pathophysiology.
Topics: Animals; Epithelial Cells; Humans; LIM-Homeodomain Proteins; Macular Degeneration; Mesenchymal Stem Cells; Rats; Retinal Degeneration; Retinal Pigment Epithelium; Retinal Pigments; Transcription Factors; Umbilical Cord
PubMed: 36096985
DOI: 10.1038/s41419-022-05199-5