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Aytekin A, Yazir Y, Duruksu G, Öztürk A. Comparison of aquaporin profile of advanced passage mesenchymal stem cells with early passage mesenchymal stem cells and determination of its effect on adipogenic differentiation efficiency. Tissue Cell 2024; 89:102448. [PMID: 38917601 DOI: 10.1016/j.tice.2024.102448] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2024] [Revised: 06/11/2024] [Accepted: 06/14/2024] [Indexed: 06/27/2024]
Abstract
OBJECTIVE Our study aimed to compare aquaporin profiles in advanced and early passage bone marrow-derived mesenchymal stem cells (BM-MSCs) and assess the impact of aquaporin changes after adipogenic differentiation. Aquaporins are crucial for stem cell survival and differentiation during their life cycle. We focused on the role of aquaporins in the cell structures of advanced and early passage stem cells. METHODS In our study, BM-MSCs were used for our objectives. Characterization of the cells was evaluated via flow cytometry using stem cell surface markers. The characterized BM-MSCs were divided into control and differentiation groups at passages 3 (P3) and 8 (P8). AQP1, AQP3, AQP7, AQP9, and AQP10 expression levels on days 0, 1, 3, 7, 14, and 21 were evaluated using Real Time-PCR, ELISA, and immunofluorescence studies. RESULTS The cells were characterized by flow cytometry and confirmed to exhibit BM-MSC characteristics. At P3 and P8, differentiation was initiated, and AQP protein expression was observed to initially increase and then decrease on subsequent days. The increase in AQP protein expression at P3 occurred earlier than that at P8. Gene expression analysis demonstrated a statistically significant increase in AQP gene expression on days when AQP protein expression decreased. Moreover, statistical differences were observed between late and early passage AQP profiles. CONCLUSION Our study examined the composition of AQPs in BM-MSCs in association with cell passage, and found that AQPs play a role in the differentiation process. The connection between the AQP profile and aging might be related to differentiation capacity, which could have implications for slowing down cellular aging and developing new therapeutic approaches.
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Affiliation(s)
- Ayşegül Aytekin
- Department of Histology and Embryology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
| | - Yusufhan Yazir
- Department of Stem Cell, Institute of Health Sciences, Kocaeli University, Kocaeli, Turkey; Center for Stem Cell and Gene Therapies Research and Practice, Kocaeli University, Kocaeli, Turkey; Department of Histology and Embryology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey.
| | - Gökhan Duruksu
- Department of Stem Cell, Institute of Health Sciences, Kocaeli University, Kocaeli, Turkey; Center for Stem Cell and Gene Therapies Research and Practice, Kocaeli University, Kocaeli, Turkey
| | - Ahmet Öztürk
- Department of Stem Cell, Institute of Health Sciences, Kocaeli University, Kocaeli, Turkey; Center for Stem Cell and Gene Therapies Research and Practice, Kocaeli University, Kocaeli, Turkey; Department of Histology and Embryology, Faculty of Medicine, Kocaeli University, Kocaeli, Turkey
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Smith IM, Stroka KM. The multifaceted role of aquaporins in physiological cell migration. Am J Physiol Cell Physiol 2023; 325:C208-C223. [PMID: 37246634 PMCID: PMC10312321 DOI: 10.1152/ajpcell.00502.2022] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Revised: 05/12/2023] [Accepted: 05/22/2023] [Indexed: 05/30/2023]
Abstract
Cell migration is an essential process that underlies many physiological processes, including the immune response, organogenesis in the embryo, and angiogenesis, as well as pathological processes such as cancer metastasis. Cells have at their disposal a variety of migratory behaviors and mechanisms that seem to be specific to cell type and the microenvironment. Research over the past two decades has elucidated the water channel protein family of aquaporins (AQPs) as a regulator of many cell migration-related processes, from physical phenomena to biological signaling pathways. The roles that AQPs play in cell migration are both cell type- and isoform-specific; thus, a large swath of information has accumulated as researchers seek to identify the responses across these distinct variables. There does not seem to be a universal role that AQPs play in cell migration; the complex interplay between AQPs and cell volume management, signaling pathway activation, and in a few identified circumstances, gene expression regulation, has shown the intricate, and perhaps paradoxical, role of AQPs in cell migration. The objective of this review is to provide an organized and integrated collection of recent work that has elucidated the many mechanisms by which AQPs regulate cell migration.NEW & NOTEWORTHY Research has elucidated the water channel protein family of aquaporins (AQPs) as a regulator of many cell migration-related processes, from physical phenomena to biological signaling pathways. The roles that AQPs play in cell migration are both cell type- and isoform-specific; thus, a large swath of information has accumulated as researchers seek to identify the responses across these distinct variables. This review compiles insights into the recent findings linking AQPs to physiological cell migration.
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Affiliation(s)
- Ian M Smith
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States
| | - Kimberly M Stroka
- Fischell Department of Bioengineering, University of Maryland, College Park, Maryland, United States
- Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, Maryland, United States
- Biophysics Program, University of Maryland, College Park, Maryland, United States
- Center for Stem Cell Biology and Regenerative Medicine, University of Maryland, Baltimore, Maryland, United States
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Daniele E, Bosio L, Hussain NA, Ferrari B, Ferrari S, Barbaro V, McArdle B, Rassu N, Mura M, Parmeggiani F, Ponzin D. Denuded Descemet's membrane supports human embryonic stem cell-derived retinal pigment epithelial cell culture. PLoS One 2023; 18:e0281404. [PMID: 36745611 PMCID: PMC9901769 DOI: 10.1371/journal.pone.0281404] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Accepted: 01/22/2023] [Indexed: 02/07/2023] Open
Abstract
Recent clinical studies suggest that retinal pigment epithelial (RPE) cell replacement therapy may preserve vision in retinal degenerative diseases. Scaffold-based methods are being tested in ongoing clinical trials for delivering pluripotent-derived RPE cells to the back of the eye. The aim of this study was to investigate human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells survival and behaviour on a decellularized Descemet's Membrane (DM), which may be of clinical relevance in retinal transplantation. DMs were isolated from human donor corneas and treated with thermolysin. The DM surface topology and the efficiency of the denudation method were evaluated by atomic force microscope, scanning electron microscopy and histology. hESC-RPE cells were seeded onto the endothelial-side surface of decellularized DM in order to determine the potential of the membrane to support hESC-RPE cell culture, alongside maintaining their viability. Integrity of the hESC-RPE monolayer was assessed by measuring transepithelial resistance. RPE-specific gene expression and growth factors secretion were assessed to confirm maturation and functionality of the cells over the new substrate. Thermolysin treatment did not affect the integrity of the tissue, thus ensuring a reliable method to standardize the preparation of decellularized DM. 24 hours post-seeding, hESC-RPE cell attachment and initial proliferation rate over the denuded DM were higher than hESC-RPE cells cultured on tissue culture inserts. On the new matrix, hESC-RPE cells succeeded in forming an intact monolayer with mature tight junctions. The resulting cell culture showed characteristic RPE cell morphology and proper protein localization. Gene expression analysis and VEGF secretion demonstrate DM provides supportive scaffolding and inductive properties to enhance hESC-RPE cells maturation. Decellularized DM was shown to be capable of sustaining hESC-RPE cells culture, thus confirming to be potentially a suitable candidate for retinal cell therapy.
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Affiliation(s)
- Elena Daniele
- Department of Translational Medicine, University of Ferrara, Ferrara, Italy
- Veneto Eye Bank Foundation, Venice, Italy
- * E-mail:
| | | | - Noor Ahmed Hussain
- Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
| | | | | | | | - Brian McArdle
- The Eye-Bank for Sight Restoration, Inc., New York City, New York, United States of America
| | - Nicolò Rassu
- Ophthalmic Unit, Ospedale dell’Angelo, Venice, Italy
| | - Marco Mura
- Department of Translational Medicine, University of Ferrara, Ferrara, Italy
| | - Francesco Parmeggiani
- Department of Translational Medicine, University of Ferrara, Ferrara, Italy
- ERN-EYE Network - Center for Retinitis Pigmentosa of Veneto Region, Camposampiero Hospital, Padua, Italy
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Wang X, Fan W, Xu Z, Zhang Q, Li N, Li R, Wang G, He S, Li W, Liao D, Zhang Z, Shu N, Huang J, Zhao C, Hou S. SOX2-positive retinal stem cells are identified in adult human pars plicata by single-cell transcriptomic analyses. MedComm (Beijing) 2023; 4:e198. [PMID: 36582303 PMCID: PMC9790047 DOI: 10.1002/mco2.198] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2022] [Revised: 11/11/2022] [Accepted: 11/14/2022] [Indexed: 12/26/2022] Open
Abstract
Stem cell therapy is a promising strategy to rescue visual impairment caused by retinal degeneration. Previous studies have proposed controversial theories about whether in situ retinal stem cells (RSCs) are present in adult human eye tissue. Single-cell RNA sequencing (scRNA-seq) has emerged as one of the most powerful tools to reveal the heterogeneity of tissue cells. By using scRNA-seq, we explored the cell heterogeneity of different subregions of adult human eyes, including pars plicata, pars plana, retinal pigment epithelium (RPE), iris, and neural retina (NR). We identified one subpopulation expressing SRY-box transcription factor 2 (SOX2) as RSCs, which were present in the pars plicata of the adult human eye. Further analysis showed the identified subpopulation of RSCs expressed specific markers aquaporin 1 (AQP1) and tetraspanin 12 (TSPAN12). We, therefore, isolated this subpopulation using these two markers by flow sorting and found that the isolated RSCs could proliferate and differentiate into some retinal cell types, including photoreceptors, neurons, RPE cells, microglia, astrocytes, horizontal cells, bipolar cells, and ganglion cells; whereas, AQP1- TSPAN12- cells did not have this differentiation potential. In conclusion, our results showed that SOX2-positive RSCs are present in the pars plicata and may be valuable for treating human retinal diseases due to their proliferation and differentiation potential.
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Abstract
Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disorder with complete penetrance affecting the macula. This is caused by a mutation in the TIMP-3. This objective narrative review aims to provide an overview of the pathophysiology, current treatment modalities, and future perspectives. A literature search was performed using "PubMed," "Web of Science," "Scopus," "ScienceDirect," "Google Scholar," "medRxiv," and "bioRxiv." The molecular mechanisms underlying SFD are not completely understood. Novel advancements in cell culture techniques, including induced pluripotent stem cells, may enable more reliable modeling of SFD. These cell culture techniques aim to shed more light on the pathophysiology of SFD, and hopefully, this may lead to the future development of treatment strategies for SFD. Currently, no gene therapy is available. The main treatment is the use of anti-vascular endothelial growth factors (anti-VEGF) to treat secondary choroidal neovascular membrane (CNV), which is a major complication observed in this condition. If CNV is detected and treated promptly, patients with SFD have a good chance of maintaining a functional central vision. Other treatment modalities have been tried but have shown limited benefit, and therefore, have not managed to be more widely accepted. In summary, although there is no definitive cure yet, the use of anti-VEGF treatment for secondary CNV has provided the opportunity to maintain functional vision in individuals with SFD, provided CNV is detected and treated early.
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Affiliation(s)
- Georgios Tsokolas
- Medical Retina and Uveitis Fellow, Moorfields Eye Hospital, London, United Kingdom
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Li D, Zhang Q, Zhou Y, Zhu H, Li T, Du F. A novel nitidine chloride nanoparticle overcomes the stemness of CD133 +EPCAM + Huh7 hepatocellular carcinoma cells for liver cancer therapy. BMC Pharmacol Toxicol 2022; 23:48. [PMID: 35820920 PMCID: PMC9277916 DOI: 10.1186/s40360-022-00589-z] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Accepted: 07/04/2022] [Indexed: 01/15/2023] Open
Abstract
Background Stemness of CD133+EPCAM+ hepatocellular carcinoma cells ensures cancer resistance to apoptosis,which is a challenge to current liver cancer treatments. In this study, we evaluated the tumorcidal activity of a novel nanoparticle of nitidine chloride (TPGS-FA/NC, TPGS-FA: folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate, NC: nitidine chloride), against human hepatocellular carcinoma (HCC) cell line Huh7 growth in vitro and in vivo. Methods Huh7 cells were treated with TPGS-FA/NC. Cell proliferation was assessed using MTT and colony assays. The expression of cell markers and signaling proteins was detected using western blot analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Huh7 cells. TPGS-FA/NC (7.5, 15, 30, 60, 120 μg/mL) dose-dependently inhibited the proliferation of HCC cells, which associated with a reduction in AQP3 and STAT3 expression. Importantly,TPGS-FA/NC (10, 20, and 40 μg/mL) significantly reduced the EpCAM+/CD133+cell numbers, suppressed the sphere formation. The in vivo antitumor efficacy of TPGS-FA/NC was proved in Huh7 cell xenograft model in BALB/c nude mice, which were administered TPGS-FA/NC(4 mg· kg − 1· d − 1, ig) for 2 weeks. Results TPGS-FA/NC dose-dependently suppressed the AQP3/STAT3/CD133 axis in Huh7 cells. In Huh7 xenograft bearing nude mice, TPGS-FA/NC administration markedly inhibited Huh7 xenograft tumor growth . Conclusions TPGS-FA/NC inhibit HCC tumor growth through multiple mechanisms, and it may be a promising candidate drug for the clinical therapy of hepatocellular carcinoma. Supplementary Information The online version contains supplementary material available at 10.1186/s40360-022-00589-z.
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Affiliation(s)
- Danni Li
- School of Chemistry and Chemical Eengineering, Guangxi Minzu University, No.158, Da Xue Xi street, Xixiangtang District, Nanning, 530006, Guangxi Province, China.
| | - Qiying Zhang
- School of Chemistry and Chemical Eengineering, Guangxi Minzu University, No.158, Da Xue Xi street, Xixiangtang District, Nanning, 530006, Guangxi Province, China
| | - Yuzhu Zhou
- School of Chemistry and Chemical Eengineering, Guangxi Minzu University, No.158, Da Xue Xi street, Xixiangtang District, Nanning, 530006, Guangxi Province, China
| | - Hua Zhu
- College of Pharmacy, Guangxi University for Chinese Medicine, No.13 , Wu He street, Qingxiu District, Nanning, 530200, Guangxi Province, China
| | - Tong Li
- College of Pharmacy, Guangxi University for Chinese Medicine, No.13 , Wu He street, Qingxiu District, Nanning, 530200, Guangxi Province, China
| | - Fangkai Du
- School of Chemistry and Chemical Eengineering, Guangxi Minzu University, No.158, Da Xue Xi street, Xixiangtang District, Nanning, 530006, Guangxi Province, China
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Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells. Curr Issues Mol Biol 2021; 43:2124-2134. [PMID: 34940121 PMCID: PMC8929048 DOI: 10.3390/cimb43030147] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Revised: 11/14/2021] [Accepted: 11/23/2021] [Indexed: 12/21/2022] Open
Abstract
Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.
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Zannetti A, Benga G, Brunetti A, Napolitano F, Avallone L, Pelagalli A. Role of Aquaporins in the Physiological Functions of Mesenchymal Stem Cells. Cells 2020; 9:2678. [PMID: 33322145 PMCID: PMC7763964 DOI: 10.3390/cells9122678] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Revised: 12/09/2020] [Accepted: 12/11/2020] [Indexed: 12/13/2022] Open
Abstract
Aquaporins (AQPs) are a family of membrane water channel proteins that control osmotically-driven water transport across cell membranes. Recent studies have focused on the assessment of fluid flux regulation in relation to the biological processes that maintain mesenchymal stem cell (MSC) physiology. In particular, AQPs seem to regulate MSC proliferation through rapid regulation of the cell volume. Furthermore, several reports have shown that AQPs play a crucial role in modulating MSC attachment to the extracellular matrix, their spread, and migration. Shedding light on how AQPs are able to regulate MSC physiological functions can increase our knowledge of their biological behaviours and improve their application in regenerative and reparative medicine.
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Affiliation(s)
- Antonella Zannetti
- Institute of Biostructure and Bioimaging, CNR, Via T. De Amicis 95, 80145 Naples, Italy;
| | - Gheorghe Benga
- Romanian Academy, Cluj-Napoca Branch, Strada Republicii 9, 400015 Cluj-Napoca, Romania;
| | - Arturo Brunetti
- Department of Advanced Biomedical Sciences, University of Naples Federico II, via Pansini 5, 80131 Naples, Italy;
| | - Francesco Napolitano
- Department of Veterinary Medicine and Animal Production, University of Naples Federico II, via Veterinaria 1, 80137 Naples, Italy; (F.N.); (L.A.)
- CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Naples, Italy
| | - Luigi Avallone
- Department of Veterinary Medicine and Animal Production, University of Naples Federico II, via Veterinaria 1, 80137 Naples, Italy; (F.N.); (L.A.)
| | - Alessandra Pelagalli
- Institute of Biostructure and Bioimaging, CNR, Via T. De Amicis 95, 80145 Naples, Italy;
- Department of Advanced Biomedical Sciences, University of Naples Federico II, via Pansini 5, 80131 Naples, Italy;
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Fields MA, Del Priore LV, Adelman RA, Rizzolo LJ. Interactions of the choroid, Bruch's membrane, retinal pigment epithelium, and neurosensory retina collaborate to form the outer blood-retinal-barrier. Prog Retin Eye Res 2019; 76:100803. [PMID: 31704339 DOI: 10.1016/j.preteyeres.2019.100803] [Citation(s) in RCA: 88] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2019] [Revised: 10/26/2019] [Accepted: 10/28/2019] [Indexed: 01/10/2023]
Abstract
The three interacting components of the outer blood-retinal barrier are the retinal pigment epithelium (RPE), choriocapillaris, and Bruch's membrane, the extracellular matrix that lies between them. Although previously reviewed independently, this review integrates these components into a more wholistic view of the barrier and discusses reconstitution models to explore the interactions among them. After updating our understanding of each component's contribution to barrier function, we discuss recent efforts to examine how the components interact. Recent studies demonstrate that claudin-19 regulates multiple aspects of RPE's barrier function and identifies a barrier function whereby mutations of claudin-19 affect retinal development. Co-culture approaches to reconstitute components of the outer blood-retinal barrier are beginning to reveal two-way interactions between the RPE and choriocapillaris. These interactions affect barrier function and the composition of the intervening Bruch's membrane. Normal or disease models of Bruch's membrane, reconstituted with healthy or diseased RPE, demonstrate adverse effects of diseased matrix on RPE metabolism. A stumbling block for reconstitution studies is the substrates typically used to culture cells are inadequate substitutes for Bruch's membrane. Together with human stem cells, the alternative substrates that have been designed offer an opportunity to engineer second-generation culture models of the outer blood-retinal barrier.
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Affiliation(s)
- Mark A Fields
- Department of Ophthalmology and Visual Science, Yale University School of Medicine, PO Box 208061, New Haven, CT, 06520-8061, USA
| | - Lucian V Del Priore
- Department of Ophthalmology and Visual Science, Yale University School of Medicine, PO Box 208061, New Haven, CT, 06520-8061, USA
| | - Ron A Adelman
- Department of Ophthalmology and Visual Science, Yale University School of Medicine, PO Box 208061, New Haven, CT, 06520-8061, USA
| | - Lawrence J Rizzolo
- Department of Ophthalmology and Visual Science, Yale University School of Medicine, PO Box 208061, New Haven, CT, 06520-8061, USA; Department of Surgery, Yale University School of Medicine, PO Box 208062, New Haven, CT, 06520-8062, USA.
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Ilmarinen T, Thieltges F, Hongisto H, Juuti‐Uusitalo K, Koistinen A, Kaarniranta K, Brinken R, Braun N, Holz FG, Skottman H, Stanzel BV. Survival and functionality of xeno-free human embryonic stem cell-derived retinal pigment epithelial cells on polyester substrate after transplantation in rabbits. Acta Ophthalmol 2019; 97:e688-e699. [PMID: 30593729 DOI: 10.1111/aos.14004] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2017] [Accepted: 11/21/2018] [Indexed: 01/08/2023]
Abstract
PURPOSE To study immunogenic properties of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and to evaluate subretinal xenotransplantation of hESC-RPE on porous polyethylene terephthalate (PET) in rabbits. METHODS Human ESC-RPE cells were characterized by morphology, transepithelial electrical resistance (TER), protein expression and photoreceptor outer segment phagocytosis in vitro. Expression of major histocompatibility complex (MHC) proteins was assessed in conventionally or xeno-free produced hESC-RPE ± interferon-gamma (IFN-γ) stimulation (n = 1). Xeno-free hESC-RPE on PET with TER < 200 Ω·cm2 > or PET alone were transplanted into 18 rabbits with short-term triamcinolone ± extended tacrolimus immunosuppression. Rabbits were monitored by spectral domain optical coherence tomography. After 4 weeks, the eyes were processed for histology and transmission electron microscopy. RESULTS Upon in vitro IFN-γ stimulation, xeno-free hESC-RPE expressed lower level of MHC-II proteins compared to the conventional cells. Outer nuclear layer (ONL) atrophy was observed over the graft in most cases 4 weeks post-transplantation. In 3/4 animals with high TER hESC-RPE, but only in 1/3 animals with low TER hESC-RPE, ONL atrophy was observed already within 1 week. Retinal cell infiltrations were more frequent in animals with high TER hESC-RPE. However, the difference was not statistically significant. In three animals, preservation of ONL was observed. Weekly intravitreal tacrolimus did not affect ONL preservation. In all animals, hESC-RPE cells survived for 4 weeks, but without tacrolimus, enlarged vacuoles accumulated in hESC-RPE (n = 1). CONCLUSIONS Xenografted xeno-free hESC-RPE monolayers can survive and retain some functionality for 4 weeks following short-term immunosuppression. The preliminary findings of this study suggest that further investigations to improve transplantation success of hESC-RPE xenografts in rabbits should be addressed especially toward the roles of hESC-RPE maturation stage and extended intravitreal immunosuppression.
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Affiliation(s)
- Tanja Ilmarinen
- BioMediTech Institute Faculty of Medicine and Life Sciences University of Tampere Tampere Finland
| | | | - Heidi Hongisto
- BioMediTech Institute Faculty of Medicine and Life Sciences University of Tampere Tampere Finland
- Department of Ophthalmology Institute of Clinical Medicine University of Eastern Finland Kuopio Finland
| | - Kati Juuti‐Uusitalo
- BioMediTech Institute Faculty of Medicine and Life Sciences University of Tampere Tampere Finland
| | | | - Kai Kaarniranta
- Department of Ophthalmology Institute of Clinical Medicine University of Eastern Finland Kuopio Finland
- Department of Ophthalmology Kuopio University Hospital Kuopio Finland
| | - Ralf Brinken
- Department of Ophthalmology University of Bonn Bonn Germany
| | | | - Frank G. Holz
- Department of Ophthalmology University of Bonn Bonn Germany
| | - Heli Skottman
- BioMediTech Institute Faculty of Medicine and Life Sciences University of Tampere Tampere Finland
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Wang Y, Wu G, Fu X, Xu S, Wang T, Zhang Q, Yang Y. Aquaporin 3 maintains the stemness of CD133+ hepatocellular carcinoma cells by activating STAT3. Cell Death Dis 2019; 10:465. [PMID: 31197130 PMCID: PMC6565673 DOI: 10.1038/s41419-019-1712-0] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 05/28/2019] [Accepted: 05/29/2019] [Indexed: 02/08/2023]
Abstract
An increasing interest in liver cancer stemness arises owing to its aggressive behavior and poor prognosis. CD133, a widely known liver cancer stem cell marker, plays critical roles in the maintenance of liver cancer stemness. Thus, exploring the regulatory mechanism of CD133 expression is significant. In the present study, we proved the carcinogenesis roles of aquaporin 3 (AQP3) in hepatocellular carcinoma (HCC) and demonstrated that AQP3 promotes the stem cell-like properties of hepatoma cells by regulating CD133 expression. In addition, AQP3 promoted the stimulation and nuclear translocation of signal transducer and activator of transcription 3 (STAT3) with a subsequent increase in the level of CD133 promoter-acetylated histone H3. This phenomenon accelerated CD133 transcription. Next, whether AQP3 acted as an oncogenic gene in HCC and maintained the stemness of CD133+ hepatoma cells were elucidated; also, a novel mechanism underlying the AQP3/STAT3/CD133 pathway in HCC was deduced.
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Affiliation(s)
- Yawei Wang
- Department of Geriatric Surgery, The First Affiliated Hospital of China Medical University, Shenyang, PR China
| | - Gang Wu
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of China Medical University, Shenyang, PR China.
| | - Xueyan Fu
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of China Medical University, Shenyang, PR China
| | - Shaolin Xu
- Department of Hepatobiliary Surgery, The First Affiliated Hospital of China Medical University, Shenyang, PR China
| | - Tianlong Wang
- Department of Geriatric Surgery, The First Affiliated Hospital of China Medical University, Shenyang, PR China
| | - Qi Zhang
- Department of Geriatric Surgery, The First Affiliated Hospital of China Medical University, Shenyang, PR China
| | - Ye Yang
- Department of Geriatric Surgery, The First Affiliated Hospital of China Medical University, Shenyang, PR China
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Dvoriashyna M, Foss AJ, Gaffney EA, Jensen OE, Repetto R. Osmotic and electroosmotic fluid transport across the retinal pigment epithelium: A mathematical model. J Theor Biol 2018; 456:233-248. [DOI: 10.1016/j.jtbi.2018.08.009] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2018] [Revised: 08/03/2018] [Accepted: 08/06/2018] [Indexed: 01/26/2023]
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Chang YK, Hwang JS, Chung TY, Shin YJ. SOX2 Activation Using CRISPR/dCas9 Promotes Wound Healing in Corneal Endothelial Cells. Stem Cells 2018; 36:1851-1862. [DOI: 10.1002/stem.2915] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2018] [Revised: 08/12/2018] [Accepted: 08/18/2018] [Indexed: 01/06/2023]
Affiliation(s)
- Yoon Kyung Chang
- Department of Ophthalmology; Hallym University Medical Center, Hallym University College of Medicine; Seoul Republic of Korea
| | - Jin Sun Hwang
- Department of Ophthalmology; Hallym University Medical Center, Hallym University College of Medicine; Seoul Republic of Korea
| | - Tae-Young Chung
- Department of Ophthalmology; Samsung Medical Center, Sungkyunkwan University; Seoul Republic of Korea
| | - Young Joo Shin
- Department of Ophthalmology; Hallym University Medical Center, Hallym University College of Medicine; Seoul Republic of Korea
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14
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Potential Interplay between Hyperosmolarity and Inflammation on Retinal Pigmented Epithelium in Pathogenesis of Diabetic Retinopathy. Int J Mol Sci 2018; 19:ijms19041056. [PMID: 29614818 PMCID: PMC5979527 DOI: 10.3390/ijms19041056] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2018] [Revised: 03/21/2018] [Accepted: 03/30/2018] [Indexed: 12/15/2022] Open
Abstract
Diabetic retinopathy is a frequent eyesight threatening complication of type 1 and type 2 diabetes. Under physiological conditions, the inner and the outer blood-retinal barriers protect the retina by regulating ion, protein, and water flux into and out of the retina. During diabetic retinopathy, many factors, including inflammation, contribute to the rupture of the inner and/or the outer blood-retinal barrier. This rupture leads the development of macular edema, a foremost cause of sight loss among diabetic patients. Under these conditions, it has been speculated that retinal pigmented epithelial cells, that constitute the outer blood-retinal barrier, may be subjected to hyperosmolar stress resulting from different mechanisms. Herein, we review the possible origins and consequences of hyperosmolar stress on retinal pigmented epithelial cells during diabetic retinopathy, with a special focus on the intimate interplay between inflammation and hyperosmolar stress, as well as the current and forthcoming new pharmacotherapies for the treatment of such condition.
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15
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Graziano ACE, Avola R, Pannuzzo G, Cardile V. Aquaporin1 and 3 modification as a result of chondrogenic differentiation of human mesenchymal stem cell. J Cell Physiol 2018; 233:2279-2291. [PMID: 28708257 DOI: 10.1002/jcp.26100] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2017] [Accepted: 07/13/2017] [Indexed: 12/15/2022]
Abstract
Chondrocytes are cells of articular cartilage particularly sensitive to water transport and ionic and osmotic changes from extracellular environment and responsible for the production of the synovial fluid. Aquaporins (AQPs) are a family of water and small solute transport channel proteins identified in several tissues, involved in physiological pathways and in manifold human diseases. In a recent period, AQP1 and 3 seem to have a role in metabolic water regulation in articular cartilage of load bearing joints. The aim of this study was to examine the levels of AQP1 and 3 during the chondrogenic differentiation of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT). For the determination of chondrogenic markers and AQPs levels, glycosaminoglycans (GAGs) quantification, immunocytochemistry, RT-PCR, and Western blot were used after 0, 7, 14, 21, and 28 days from the start of differentiation. At 21 days, chondrocytes derived from AT-MSCs were able to produce augmented content of GAGs and significant quantity of SOX-9, lubricin, aggrecan, and collagen type II, suggesting hyaline cartilage formation, in combination with an increase of AQP3 and AQP1. However, while AQP1 level decreased after 21 days; AQP3 reached higher values at 28 days. The expression of AQP1 and 3 is a manifestation of physiological adaptation of functionally mature chondrocytes able to respond to the change of their internal environment influenced by extracellular matrix. The alteration or loss of expression of AQP1 and 3 could contribute to destruction of chondrocytes and to development of cartilage damage.
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Affiliation(s)
- Adriana C E Graziano
- Department of Biomedical and Biotechnological Science, Section of Physiology, University of Catania, Catania, Italy
| | - Rosanna Avola
- Department of Biomedical and Biotechnological Science, Section of Physiology, University of Catania, Catania, Italy
| | - Giovanna Pannuzzo
- Department of Biomedical and Biotechnological Science, Section of Physiology, University of Catania, Catania, Italy
| | - Venera Cardile
- Department of Biomedical and Biotechnological Science, Section of Physiology, University of Catania, Catania, Italy
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16
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Mechanisms of macular edema: Beyond the surface. Prog Retin Eye Res 2017; 63:20-68. [PMID: 29126927 DOI: 10.1016/j.preteyeres.2017.10.006] [Citation(s) in RCA: 413] [Impact Index Per Article: 51.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2017] [Revised: 10/24/2017] [Accepted: 10/31/2017] [Indexed: 02/07/2023]
Abstract
Macular edema consists of intra- or subretinal fluid accumulation in the macular region. It occurs during the course of numerous retinal disorders and can cause severe impairment of central vision. Major causes of macular edema include diabetes, branch and central retinal vein occlusion, choroidal neovascularization, posterior uveitis, postoperative inflammation and central serous chorioretinopathy. The healthy retina is maintained in a relatively dehydrated, transparent state compatible with optimal light transmission by multiple active and passive systems. Fluid accumulation results from an imbalance between processes governing fluid entry and exit, and is driven by Starling equation when inner or outer blood-retinal barriers are disrupted. The multiple and intricate mechanisms involved in retinal hydro-ionic homeostasis, their molecular and cellular basis, and how their deregulation lead to retinal edema, are addressed in this review. Analyzing the distribution of junction proteins and water channels in the human macula, several hypotheses are raised to explain why edema forms specifically in the macular region. "Pure" clinical phenotypes of macular edema, that result presumably from a single causative mechanism, are detailed. Finally, diabetic macular edema is investigated, as a complex multifactorial pathogenic example. This comprehensive review on the current understanding of macular edema and its mechanisms opens perspectives to identify new preventive and therapeutic strategies for this sight-threatening condition.
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17
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Skottman H, Muranen J, Lähdekorpi H, Pajula E, Mäkelä K, Koivusalo L, Koistinen A, Uusitalo H, Kaarniranta K, Juuti-Uusitalo K. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells. Exp Cell Res 2017; 359:101-111. [DOI: 10.1016/j.yexcr.2017.08.004] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2017] [Revised: 07/31/2017] [Accepted: 08/02/2017] [Indexed: 10/19/2022]
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18
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Sorsby fundus dystrophy - A review of pathology and disease mechanisms. Exp Eye Res 2017; 165:35-46. [PMID: 28847738 DOI: 10.1016/j.exer.2017.08.014] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2017] [Revised: 08/23/2017] [Accepted: 08/23/2017] [Indexed: 01/29/2023]
Abstract
Sorsby fundus dystrophy (SFD) is an autosomal dominant macular dystrophy with an estimated prevalence of 1 in 220,000 and an onset of disease around the 4th to 6th decade of life. Similar to age-related macular degeneration (AMD), ophthalmoscopy reveals accumulation of protein/lipid deposits under the retinal pigment epithelium (RPE), referred to as drusen, in the eyes of patients with SFD. SFD is caused by variants in the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which has been found in drusen-like deposits of SFD patients. TIMP3 is constitutively expressed by RPE cells and, in healthy eyes, resides in Bruch's membrane. Most SFD-associated TIMP3 variants involve the gain or loss of a cysteine residue. This suggests the protein aberrantly forms intermolecular disulphide bonds, resulting in the formation of TIMP3 dimers. It has been demonstrated that SFD-associated TIMP3 variants are more resistant to turnover, which is thought to be a result of dimerisation and thought to explain the accumulation of TIMP3 in drusen-like deposits at the level of Bruch's membrane. An important function of TIMP3 within the outer retina is to regulate the thickness of Bruch's membrane. TIMP3 performs this function by inhibiting the activity of matrix metalloproteinases (MMPs), which have the function of catalysing breakdown of the extracellular matrix. TIMP3 has an additional function to inhibit vascular endothelial growth factor (VEGF) signalling and thereby to inhibit angiogenesis. However, it is unclear whether SFD-associated TIMP3 variant proteins retain these functions. In this review, we discuss the current understanding of the potential mechanisms underlying development of SFD and summarise all known SFD-associated TIMP3 variants. Cell culture models provide an invaluable way to study disease and identify potential treatments. These allow a greater understanding of RPE physiology and pathophysiology, including the ability to study the blood-retinal barrier as well as other RPE functions such as phagocytosis of photoreceptor outer segments. This review describes some examples of such recent in vitro studies and how they might provide new insights into degenerative diseases like SFD. Thus far, most studies on SFD have been performed using ARPE-19 cells or other, less suitable, cell-types. Now, induced pluripotent stem cell (iPSC) technologies allow the possibility to non-invasively collect somatic cells, such as dermal fibroblast cells and reprogram those to produce iPSCs. Subsequent differentiation of iPSCs can generate patient-derived RPE cells that carry the same disease-associated variant as RPE cells in the eyes of the patient. Use of these patient-derived RPE cells in novel cell culture systems should increase our understanding of how SFD and similar macular dystrophies develop.
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19
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Hongisto H, Jylhä A, Nättinen J, Rieck J, Ilmarinen T, Veréb Z, Aapola U, Beuerman R, Petrovski G, Uusitalo H, Skottman H. Comparative proteomic analysis of human embryonic stem cell-derived and primary human retinal pigment epithelium. Sci Rep 2017; 7:6016. [PMID: 28729539 PMCID: PMC5519552 DOI: 10.1038/s41598-017-06233-9] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2016] [Accepted: 06/12/2017] [Indexed: 01/28/2023] Open
Abstract
Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) provide an unlimited cell source for retinal cell replacement therapies. Clinical trials using hESC-RPE to treat diseases such as age-related macular degeneration (AMD) are currently underway. Human ESC-RPE cells have been thoroughly characterized at the gene level but their protein expression profile has not been studied at larger scale. In this study, proteomic analysis was used to compare hESC-RPE cells differentiated from two independent hESC lines, to primary human RPE (hRPE) using Isobaric tags for relative quantitation (iTRAQ). 1041 common proteins were present in both hESC-RPE cells and native hRPE with majority of the proteins similarly regulated. The hESC-RPE proteome reflected that of normal hRPE with a large number of metabolic, mitochondrial, cytoskeletal, and transport proteins expressed. No signs of increased stress, apoptosis, immune response, proliferation, or retinal degeneration related changes were noted in hESC-RPE, while important RPE specific proteins involved in key RPE functions such as visual cycle and phagocytosis, could be detected in the hESC-RPE. Overall, the results indicated that the proteome of the hESC-RPE cells closely resembled that of their native counterparts.
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Affiliation(s)
- Heidi Hongisto
- BioMediTech Institute, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.
| | - Antti Jylhä
- Department of Ophthalmology, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
| | - Janika Nättinen
- BioMediTech Institute, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.,Department of Ophthalmology, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
| | - Jochen Rieck
- BioMediTech Institute, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
| | - Tanja Ilmarinen
- BioMediTech Institute, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
| | - Zoltán Veréb
- Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary
| | - Ulla Aapola
- Department of Ophthalmology, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
| | - Roger Beuerman
- Department of Ophthalmology, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.,Singapore Eye Research Institute and Duke-NUS School of Medicine, Singapore, Singapore
| | - Goran Petrovski
- Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.,Center for Eye Research, Department of Ophthalmology, Oslo University Hospital and University of Oslo, Oslo, Norway
| | - Hannu Uusitalo
- Department of Ophthalmology, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland.,Tampere University Hospital Eye Center, University of Tampere, Tampere, Finland
| | - Heli Skottman
- BioMediTech Institute, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
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20
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Tarchick MJ, Bassiri P, Rohwer RM, Samuels IS. Early Functional and Morphologic Abnormalities in the Diabetic Nyxnob Mouse Retina. Invest Ophthalmol Vis Sci 2017; 57:3496-508. [PMID: 27367517 PMCID: PMC4961059 DOI: 10.1167/iovs.15-18775] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Purpose The electroretinogram c-wave is generated by the summation of the positive polarity hyperpolarization of the apical RPE membrane and a negative polarity slow PIII response of Müller glia cells. Therefore, the c-wave reduction noted in prior studies of mouse models of diabetes could reflect a reduction in the RPE component or an increase in slow PIII. The present study used a genetic approach to distinguish between these two alternatives. Methods Nyxnob mice lack the ERG b-wave, revealing the early phase of slow PIII. To visualize changes in slow PIII due to diabetes, Nyxnob mice were given streptozotocin (STZ) injections to induce diabetes or received vehicle as a control. After 1, 2, and 4 weeks of sustained hyperglycemia (>250 mg/dL), standard strobe flash ERG and dc-ERG testing were conducted. Histological analysis of the retina was performed. Results A reduced c-wave was noted at the 1 week time point, and persisted at later time points. In comparison, slow PIII amplitudes were unaffected after 1 week of hyperglycemia, but were significantly reduced in STZ mice at the 2-week time point. The decrease in amplitude occurred before any identifiable decrease to the a-wave. At the later time point, the a-wave became involved, although the slow PIII reductions were more pronounced. Morphological abnormalities in the RPE, including increased thickness and altered melanosome distribution, were identified in diabetic animals. Conclusions Because the c-wave and slow PIII were both reduced, these results demonstrated that diabetes-induced reductions to the c-wave cannot be attributed to an early increase in the Müller glia-derived potassium conductance. Furthermore, because the a-wave, slow PIII and c-wave reductions were not equivalent, and varied in their onset, the reductions cannot reflect the same mechanism, such as a change in membrane resistance. The presence of small changes to RPE architecture indicate that the c-wave reductions present in diabetic mice likely represents a primary change in the RPE induced by hyperglycemia.
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Affiliation(s)
- Matthew J Tarchick
- Louis Stokes Cleveland VA Medical Center, Cleveland, Ohio, United States 2Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
| | - Parastoo Bassiri
- Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
| | - Rebecca M Rohwer
- Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
| | - Ivy S Samuels
- Louis Stokes Cleveland VA Medical Center, Cleveland, Ohio, United States 2Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
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21
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Autophagy Regulates Proteasome Inhibitor-Induced Pigmentation in Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells. Int J Mol Sci 2017; 18:ijms18051089. [PMID: 28534814 PMCID: PMC5454998 DOI: 10.3390/ijms18051089] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2017] [Revised: 05/11/2017] [Accepted: 05/12/2017] [Indexed: 02/06/2023] Open
Abstract
The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes.
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22
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Xia T, Rizzolo LJ. Effects of diabetic retinopathy on the barrier functions of the retinal pigment epithelium. Vision Res 2017; 139:72-81. [PMID: 28347688 DOI: 10.1016/j.visres.2017.02.006] [Citation(s) in RCA: 83] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2016] [Accepted: 02/28/2017] [Indexed: 02/06/2023]
Abstract
Diabetic retinopathy is a debilitating microvascular complication of diabetes mellitus. A rich literature describes the breakdown of retinal endothelial cells and the inner blood-retinal barrier, but the effects of diabetes on the retinal pigment epithelium (RPE) has received much less attention. RPE lies between the choroid and neurosensory retina to form the outer blood-retinal barrier. RPE's specialized and dynamic barrier functions are crucial for maintaining retinal health. RPE barrier functions include a collection of interrelated structures and activities that regulate the transepithelial movement of solutes, including: diffusion through the paracellular spaces, facilitated diffusion through the cells, active transport, receptor-mediated and bulk phase transcytosis, and metabolic processing of solutes in transit. In the later stages of diabetic retinopathy, the tight junctions that regulate the paracellular space begin to disassemble, but there are earlier effects on the other aspects of RPE barrier function, particularly active transport and metabolic processing. With advanced understanding of RPE-specific barrier functions, and more in vivo-like culture models, the time is ripe for revisiting experiments in the literature to resolve controversies and extend our understanding of how diabetes affects the outer blood-retinal barrier.
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Affiliation(s)
- Tina Xia
- Departments of Surgery and Ophthalmology and Visual Science, Yale University School of Medicine, PO Box 208062, New Haven, CT 06520-8062, USA.
| | - Lawrence J Rizzolo
- Departments of Surgery and Ophthalmology and Visual Science, Yale University School of Medicine, PO Box 208062, New Haven, CT 06520-8062, USA.
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23
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Sorkio A, Haimi S, Verdoold V, Juuti-Uusitalo K, Grijpma D, Skottman H. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells. J Tissue Eng Regen Med 2017; 11:3134-3144. [DOI: 10.1002/term.2221] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2015] [Revised: 03/15/2016] [Accepted: 04/19/2016] [Indexed: 12/28/2022]
Affiliation(s)
- Anni Sorkio
- BioMediTech; University of Tampere; Tampere Finland
| | - Suvi Haimi
- BioMediTech; University of Tampere; Tampere Finland
- MIRA Institute for Biomedical Engineering and Technical Medicine and Department of Biomaterials Science and Technology; University of Twente; Enschede The Netherlands
| | - Vincent Verdoold
- MIRA Institute for Biomedical Engineering and Technical Medicine and Department of Biomaterials Science and Technology; University of Twente; Enschede The Netherlands
| | | | - Dirk Grijpma
- MIRA Institute for Biomedical Engineering and Technical Medicine and Department of Biomaterials Science and Technology; University of Twente; Enschede The Netherlands
- Department of Biomedical Engineering; University of Groningen, University Medical Centre Groningen; Groningen The Netherlands
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Nanni L, Paci M, Caetano dos Santos FL, Skottman H, Juuti-Uusitalo K, Hyttinen J. Texture Descriptors Ensembles Enable Image-Based Classification of Maturation of Human Stem Cell-Derived Retinal Pigmented Epithelium. PLoS One 2016; 11:e0149399. [PMID: 26895509 PMCID: PMC4760937 DOI: 10.1371/journal.pone.0149399] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Accepted: 02/01/2016] [Indexed: 12/02/2022] Open
Abstract
Aims A fast, non-invasive and observer-independent method to analyze the homogeneity and maturity of human pluripotent stem cell (hPSC) derived retinal pigment epithelial (RPE) cells is warranted to assess the suitability of hPSC-RPE cells for implantation or in vitro use. The aim of this work was to develop and validate methods to create ensembles of state-of-the-art texture descriptors and to provide a robust classification tool to separate three different maturation stages of RPE cells by using phase contrast microscopy images. The same methods were also validated on a wide variety of biological image classification problems, such as histological or virus image classification. Methods For image classification we used different texture descriptors, descriptor ensembles and preprocessing techniques. Also, three new methods were tested. The first approach was an ensemble of preprocessing methods, to create an additional set of images. The second was the region-based approach, where saliency detection and wavelet decomposition divide each image in two different regions, from which features were extracted through different descriptors. The third method was an ensemble of Binarized Statistical Image Features, based on different sizes and thresholds. A Support Vector Machine (SVM) was trained for each descriptor histogram and the set of SVMs combined by sum rule. The accuracy of the computer vision tool was verified in classifying the hPSC-RPE cell maturation level. Dataset and Results The RPE dataset contains 1862 subwindows from 195 phase contrast images. The final descriptor ensemble outperformed the most recent stand-alone texture descriptors, obtaining, for the RPE dataset, an area under ROC curve (AUC) of 86.49% with the 10-fold cross validation and 91.98% with the leave-one-image-out protocol. The generality of the three proposed approaches was ascertained with 10 more biological image datasets, obtaining an average AUC greater than 97%. Conclusions Here we showed that the developed ensembles of texture descriptors are able to classify the RPE cell maturation stage. Moreover, we proved that preprocessing and region-based decomposition improves many descriptors’ accuracy in biological dataset classification. Finally, we built the first public dataset of stem cell-derived RPE cells, which is publicly available to the scientific community for classification studies. The proposed tool is available at https://www.dei.unipd.it/node/2357 and the RPE dataset at http://www.biomeditech.fi/data/RPE_dataset/. Both are available at https://figshare.com/s/d6fb591f1beb4f8efa6f.
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Affiliation(s)
- Loris Nanni
- Department of Information Engineering, University of Padua, Padua, Italy
- * E-mail: (LN); (MP)
| | - Michelangelo Paci
- Department of Electronics and Communications Engineering, Tampere University of Technology, BioMediTech, Tampere, Finland
- * E-mail: (LN); (MP)
| | | | - Heli Skottman
- University of Tampere, BioMediTech, Tampere, Finland
| | | | - Jari Hyttinen
- Department of Electronics and Communications Engineering, Tampere University of Technology, BioMediTech, Tampere, Finland
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25
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Sorkio A, Porter PJ, Juuti-Uusitalo K, Meenan BJ, Skottman H, Burke GA. Surface Modified Biodegradable Electrospun Membranes as a Carrier for Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells. Tissue Eng Part A 2015; 21:2301-14. [PMID: 25946229 DOI: 10.1089/ten.tea.2014.0640] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells are currently undergoing clinical trials to treat retinal degenerative diseases. Transplantation of hESC-RPE cells in conjuction with a supportive biomaterial carrier holds great potential as a future treatment for retinal degeneration. However, there has been no such biodegradable material that could support the growth and maturation of hESC-RPE cells so far. The primary aim of this work was to create a thin porous poly (L-lactide-co-caprolactone) (PLCL) membrane that could promote attachment, proliferation, and maturation of the hESC-RPE cells in serum-free culture conditions. The PLCL membranes were modified by atmospheric pressure plasma processing and coated with collagen IV to enhance cell growth and maturation. Permeability of the membranes was analyzed with an Ussing chamber system. Analysis with scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy demonstrated that plasma surface treatment augments the surface properties of the membrane, which enhances the binding and conformation of the protein. Cell proliferation assays, reverse transcription-polymerase chain reaction, indirect immunofluoresence staining, trans-epithelial electrical resistance measurements, and in vitro phagocytosis assay clearly demonstrated that the plasma treated PLCL membranes supported the adherence, proliferation, maturation and functionality of hESC-RPE cells in serum-free culture conditions. Here, we report for the first time, how PLCL membranes can be modified with atmospheric pressure plasma processing to enable the formation of a functional hESC-RPE monolayer on a porous biodegradable substrate, which have a potential as a tissue-engineered construct for regenerative retinal repair applications.
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Affiliation(s)
- Anni Sorkio
- 1 BioMediTech, University of Tampere , Tampere, Finland
| | - Patrick J Porter
- 2 Nanotechnology and Integrated Bioengineering Centre (NIBEC), School of Engineering, University of Ulster , Newtownabbey, Northern Ireland
| | | | - Brian J Meenan
- 2 Nanotechnology and Integrated Bioengineering Centre (NIBEC), School of Engineering, University of Ulster , Newtownabbey, Northern Ireland
| | - Heli Skottman
- 1 BioMediTech, University of Tampere , Tampere, Finland
| | - George A Burke
- 2 Nanotechnology and Integrated Bioengineering Centre (NIBEC), School of Engineering, University of Ulster , Newtownabbey, Northern Ireland
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Alonso-Alonso ML, Srivastava GK. Current focus of stem cell application in retinal repair. World J Stem Cells 2015; 7:641-648. [PMID: 25914770 PMCID: PMC4404398 DOI: 10.4252/wjsc.v7.i3.641] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/01/2014] [Revised: 11/06/2014] [Accepted: 01/19/2015] [Indexed: 02/06/2023] Open
Abstract
The relevance of retinal diseases, both in society’s economy and in the quality of people’s life who suffer with them, has made stem cell therapy an interesting topic for research. Embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adipose derived mesenchymal stem cells (ADMSCs) are the focus in current endeavors as a source of different retinal cells, such as photoreceptors and retinal pigment epithelial cells. The aim is to apply them for cell replacement as an option for treating retinal diseases which so far are untreatable in their advanced stage. ESCs, despite the great potential for differentiation, have the dangerous risk of teratoma formation as well as ethical issues, which must be resolved before starting a clinical trial. iPSCs, like ESCs, are able to differentiate in to several types of retinal cells. However, the process to get them for personalized cell therapy has a high cost in terms of time and money. Researchers are working to resolve this since iPSCs seem to be a realistic option for treating retinal diseases. ADMSCs have the advantage that the procedures to obtain them are easier. Despite advancements in stem cell application, there are still several challenges that need to be overcome before transferring the research results to clinical application. This paper reviews recent research achievements of the applications of these three types of stem cells as well as clinical trials currently based on them.
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Sorkio AE, Vuorimaa-Laukkanen EP, Hakola HM, Liang H, Ujula TA, Valle-Delgado JJ, Österberg M, Yliperttula ML, Skottman H. Biomimetic collagen I and IV double layer Langmuir-Schaefer films as microenvironment for human pluripotent stem cell derived retinal pigment epithelial cells. Biomaterials 2015; 51:257-269. [PMID: 25771016 DOI: 10.1016/j.biomaterials.2015.02.005] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2014] [Revised: 01/26/2015] [Accepted: 02/01/2015] [Indexed: 12/11/2022]
Abstract
The environmental cues received by the cells from synthetic substrates in vitro are very different from those they receive in vivo. In this study, we applied the Langmuir-Schaefer (LS) deposition, a variant of Langmuir-Blodgett technique, to fabricate a biomimetic microenvironment mimicking the structure and organization of native Bruch's membrane for the production of the functional human embryonic stem cell derived retinal pigment epithelial (hESC-RPE) cells. Surface pressure-area isotherms were measured simultaneously with Brewster angle microscopy to investigate the self-assembly of human collagens type I and IV on air-subphase interface. Furthermore, the structure of the prepared collagen LS films was characterized with scanning electron microscopy, atomic force microscopy, surface plasmon resonance measurements and immunofluorescent staining. The integrity of hESC-RPE on double layer LS films was investigated by measuring transepithelial resistance and permeability of small molecular weight substance. Maturation and functionality of hESC-RPE cells on double layer collagen LS films was further assessed by RPE-specific gene and protein expression, growth factor secretion, and phagocytic activity. Here, we demonstrated that the prepared collagen LS films have layered structure with oriented fibers corresponding to architecture of the uppermost layers of Bruch's membrane and result in increased barrier properties and functionality of hESC-RPE cells as compared to the commonly used dip-coated controls.
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Affiliation(s)
- Anni E Sorkio
- BioMediTech, University of Tampere FM5/BMT, 33014 University of Tampere, Finland.
| | - Elina P Vuorimaa-Laukkanen
- Department of Chemistry and Bioengineering, Tampere University of Technology, Korkeakoulunkatu 8, 33720 Tampere, Finland
| | - Hanna M Hakola
- Department of Chemistry and Bioengineering, Tampere University of Technology, Korkeakoulunkatu 8, 33720 Tampere, Finland
| | - Huamin Liang
- Department of Chemistry and Bioengineering, Tampere University of Technology, Korkeakoulunkatu 8, 33720 Tampere, Finland
| | - Tiina A Ujula
- Department of Forest Products Technology, School of Chemical Technology, Aalto University, P.O. Box 16300, 00076 Aalto, Finland
| | - Juan José Valle-Delgado
- Department of Forest Products Technology, School of Chemical Technology, Aalto University, P.O. Box 16300, 00076 Aalto, Finland
| | - Monika Österberg
- Department of Forest Products Technology, School of Chemical Technology, Aalto University, P.O. Box 16300, 00076 Aalto, Finland
| | - Marjo L Yliperttula
- Division of Biopharmaceutical Sciences, Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5, 00790 Helsinki, Finland
| | - Heli Skottman
- BioMediTech, University of Tampere FM5/BMT, 33014 University of Tampere, Finland
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Lehmann GL, Benedicto I, Philp NJ, Rodriguez-Boulan E. Plasma membrane protein polarity and trafficking in RPE cells: past, present and future. Exp Eye Res 2014; 126:5-15. [PMID: 25152359 DOI: 10.1016/j.exer.2014.04.021] [Citation(s) in RCA: 74] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2013] [Revised: 04/15/2014] [Accepted: 04/24/2014] [Indexed: 10/24/2022]
Abstract
The retinal pigment epithelium (RPE) comprises a monolayer of polarized pigmented epithelial cells that is strategically interposed between the neural retina and the fenestrated choroid capillaries. The RPE performs a variety of vectorial transport functions (water, ions, metabolites, nutrients and waste products) that regulate the composition of the subretinal space and support the functions of photoreceptors (PRs) and other cells in the neural retina. To this end, RPE cells display a polarized distribution of channels, transporters and receptors in their plasma membrane (PM) that is remarkably different from that found in conventional extra-ocular epithelia, e.g. intestine, kidney, and gall bladder. This characteristic PM protein polarity of RPE cells depends on the interplay of sorting signals in the RPE PM proteins and sorting mechanisms and biosynthetic/recycling trafficking routes in the RPE cell. Although considerable progress has been made in our understanding of the RPE trafficking machinery, most available data have been obtained from immortalized RPE cell lines that only partially maintain the RPE phenotype and by extrapolation of data obtained in the prototype Madin-Darby Canine Kidney (MDCK) cell line. The increasing availability of RPE cell cultures that more closely resemble the RPE in vivo together with the advent of advanced live imaging microscopy techniques provides a platform and an opportunity to rapidly expand our understanding of how polarized protein trafficking contributes to RPE PM polarity.
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Affiliation(s)
- Guillermo L Lehmann
- Margaret Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, 1300 York Ave, New York, NY 100652, USA
| | - Ignacio Benedicto
- Margaret Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, 1300 York Ave, New York, NY 100652, USA
| | - Nancy J Philp
- Thomas Jefferson University, Department of Pathology, Anatomy, and Cell Biology, Philadelphia, PA 19107, USA.
| | - Enrique Rodriguez-Boulan
- Margaret Dyson Vision Research Institute, Department of Ophthalmology, Weill Cornell Medical College, 1300 York Ave, New York, NY 100652, USA.
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Willermain F, Libert S, Motulsky E, Salik D, Caspers L, Perret J, Delporte C. Origins and consequences of hyperosmolar stress in retinal pigmented epithelial cells. Front Physiol 2014; 5:199. [PMID: 24910616 PMCID: PMC4038854 DOI: 10.3389/fphys.2014.00199] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2014] [Accepted: 05/09/2014] [Indexed: 01/21/2023] Open
Abstract
The retinal pigmented epithelium (RPE) is composed of retinal pigmented epithelial cells joined by tight junctions and represents the outer blood-retinal barrier (BRB). The inner BRB is made of endothelial cells joined by tight junctions and glial extensions surrounding all the retinal blood vessels. One of the functions of the RPE is to maintain an osmotic transepithelial gradient created by ionic pumps and channels, avoiding paracellular flux. Under such physiological conditions, transcellular water movement follows the osmotic gradient and flows normally from the retina to the choroid through the RPE. Several diseases, such as diabetic retinopathy, are characterized by the BRB breakdown leading to leakage of solutes, proteins, and fluid from the retina and the choroid. The prevailing hypothesis explaining macular edema formation during diabetic retinopathy incriminates the inner BRB breakdown resulting in increased osmotic pressure leading in turn to massive water accumulation that can affect vision. Under these conditions, it has been hypothesized that RPE is likely to be exposed to hyperosmolar stress at its apical side. This review summarizes the origins and consequences of osmotic stress in the RPE. Ongoing and further research advances will clarify the mechanisms, at the molecular level, involved in the response of the RPE to osmotic stress and delineate potential novel therapeutic targets and tools.
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Affiliation(s)
- François Willermain
- Department of Ophthalmology, CHU Saint-Pierre and Brugmann Brussels, Belgium ; I.R.I.B.H.M, Université Libre de Bruxelles Brussels, Belgium
| | - Sarah Libert
- Department of Ophthalmology, CHU Saint-Pierre and Brugmann Brussels, Belgium ; Laboratory of Pathophysiological and Nutritional Biochemistry, Department of Biochemistry, Université Libre de Bruxelles Brussels, Belgium
| | - Elie Motulsky
- Department of Ophthalmology, CHU Saint-Pierre and Brugmann Brussels, Belgium ; Laboratory of Pathophysiological and Nutritional Biochemistry, Department of Biochemistry, Université Libre de Bruxelles Brussels, Belgium
| | - Dany Salik
- Department of Ophthalmology, CHU Saint-Pierre and Brugmann Brussels, Belgium ; Laboratory of Pathophysiological and Nutritional Biochemistry, Department of Biochemistry, Université Libre de Bruxelles Brussels, Belgium
| | - Laure Caspers
- Department of Ophthalmology, CHU Saint-Pierre and Brugmann Brussels, Belgium
| | - Jason Perret
- Laboratory of Pathophysiological and Nutritional Biochemistry, Department of Biochemistry, Université Libre de Bruxelles Brussels, Belgium
| | - Christine Delporte
- Laboratory of Pathophysiological and Nutritional Biochemistry, Department of Biochemistry, Université Libre de Bruxelles Brussels, Belgium
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Human Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium in Retinal Treatment: from Bench to Bedside. Mol Neurobiol 2014; 50:597-612. [DOI: 10.1007/s12035-014-8684-y] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2013] [Accepted: 03/17/2014] [Indexed: 01/23/2023]
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31
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Ishibashi K, Tanaka Y, Morishita Y. The role of mammalian superaquaporins inside the cell. Biochim Biophys Acta Gen Subj 2013; 1840:1507-12. [PMID: 24189537 DOI: 10.1016/j.bbagen.2013.10.039] [Citation(s) in RCA: 102] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2013] [Revised: 10/18/2013] [Accepted: 10/23/2013] [Indexed: 01/06/2023]
Abstract
BACKGROUND The mammalian two superaquaporins, AQP11 and AQP12, are present inside the cell and their null phenotypes in mice suggest their unusual functions. SCOPE OF REVIEW The surveyed literature on these superaquaporins and our unpublished data has been incorporated to speculate their roles. MAJOR CONCLUSIONS AQP11 and AQP12 have unique NPA boxes with a signature cysteine residue. Although some water permeability of AQP11 was demonstrated in liposomes and cultured cells, its permeability to glycerol is unknown. The function of AQP12 still remains to be clarified. AQP11 null mice develop polycystic kidneys following large intracellular vacuoles in the proximal tubule, which may be caused by ER stress or vesicle fusion failure. The role of AQP11 in the kidney and liver seems to alleviate the tissue damage and facilitate the recovery. Its expression in the sperm, thymus and brain suggests its potential roles in these organs in spite of the apparently normal null phenotype. Although AQP12 null mice appear normal, they suffer from severe pancreatitis, suggesting its role in the fusion of zymogen granules. GENERAL SIGNIFICANCE As many issues are unsolved, the clarification of the function and roles of the superaquaporin may lead to the identification of new roles of AQPs. This article is part of a Special Issue entitled Aquaporins.
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Affiliation(s)
- Kenichi Ishibashi
- Department of Medical Physiology, School of Pharmacy, Meiji Pharmaceutical University, Tokyo, Japan.
| | - Yasuko Tanaka
- Department of Medical Physiology, School of Pharmacy, Meiji Pharmaceutical University, Tokyo, Japan
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