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18 pages, 8826 KiB

Open AccessArticle

Towards the Identification and Characterization of Putative Adult Human Lens Epithelial Stem Cells

by Pandi Saranya, Madhu Shekhar, Aravind Haripriya, Veerappan Muthukkaruppan and Chidambaranathan Gowri Priya

Cells 2023, 12(23), 2727; https://doi.org/10.3390/cells12232727 - 29 Nov 2023

Viewed by 945

Abstract

The anterior lens epithelium has the ability to differentiate into lens fibres throughout its life. The present study aims to identify and functionally characterize the adult stem cells in the human lens epithelium. Whole mounts of lens epithelium from donor eyes (normal/cataract) were [...] Read more.

The anterior lens epithelium has the ability to differentiate into lens fibres throughout its life. The present study aims to identify and functionally characterize the adult stem cells in the human lens epithelium. Whole mounts of lens epithelium from donor eyes (normal/cataract) were immunostained for SOX2, gap junction protein alpha 1 (GJA1), PAX6, α, β and γ-crystallins, followed by a confocal analysis. The functional property of adult stem cells was analysed by their sphere forming ability using cultured lens epithelial cells from different zones. Based on marker expression, the lens epithelium was divided into four zones: the central zone, characterized by a small population of PAX6⁺, GJA1⁻, β-crystallin⁻ and γ-crystallin⁻ cells; the germinative zone, characterized by PAX6⁺, GJA1⁺, β-crystallin⁻ and γ-crystallin⁻; the transitional zone, characterized by PAX6⁺, GJA1⁺, β-crystallin⁺ and γ-crystallin⁻; and the equatorial zone, characterized by PAX6^+/−, GJA1⁺, β-crystallin⁺, and γ-crystallin⁺ cells. The putative lens epithelial stem cells identified as SOX2⁺ and GJA1 membrane expression negative cells were located only in the central zone (1.89 ± 0.84%). Compared to the other zones, a significant percentage of spheres were identified in the central zone (1.68 ± 1.04%), consistent with the location of the putative adult lens epithelial stem cells. In the cataractous lens, an absence of SOX2 expression and a significant reduction in sphere forming ability (0.33 ± 0.11%) were observed in the central zone. The above findings confirmed the presence of putative stem cells in the central zone of the adult human lens epithelium and indicated their probable association with cataract development. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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27 pages, 9440 KiB

Open AccessArticle

ATF4 May Be Essential for Adaption of the Ocular Lens to Its Avascular Environment

by Jiawen Xiang, Anthony J. Pompetti, Adam P. Faranda, Yan Wang, Samuel G. Novo, David Wan-Cheng Li and Melinda K. Duncan

Cells 2023, 12(22), 2636; https://doi.org/10.3390/cells12222636 - 16 Nov 2023

Viewed by 862

Abstract

The late embryonic mouse lens requires the transcription factor ATF4 for its survival although the underlying mechanisms were unknown. Here, RNAseq analysis revealed that E16.5 Atf4 null mouse lenses downregulate the mRNA levels of lens epithelial markers as well as known markers of [...] Read more.

The late embryonic mouse lens requires the transcription factor ATF4 for its survival although the underlying mechanisms were unknown. Here, RNAseq analysis revealed that E16.5 Atf4 null mouse lenses downregulate the mRNA levels of lens epithelial markers as well as known markers of late lens fiber cell differentiation. However, a comparison of this list of differentially expressed genes (DEGs) with other known transcriptional regulators of lens development indicated that ATF4 expression is not directly controlled by the previously described lens gene regulatory network. Pathway analysis revealed that the Atf4 DEG list was enriched in numerous genes involved in nutrient transport, amino acid biosynthesis, and tRNA charging. These changes in gene expression likely result in the observed reductions in lens free amino acid and glutathione levels, which would result in the observed low levels of extractable lens protein, finally leading to perinatal lens disintegration. These data demonstrate that ATF4, via its function in the integrated stress response, is likely to play a crucial role in mediating the adaption of the lens to the avascularity needed to maintain lens transparency. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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16 pages, 3431 KiB

Open AccessArticle

Identification of Small Molecules for Prevention of Lens Epithelium-Derived Cataract Using Zebrafish

by Kineret Taler, Nour Zatari, Mohammad Iqbal Lone, Shahar Rotem-Bamberger and Adi Inbal

Cells 2023, 12(21), 2540; https://doi.org/10.3390/cells12212540 - 29 Oct 2023

Cited by 1 | Viewed by 954

Abstract

Cataract is the leading cause of blindness worldwide. It can be treated by surgery, whereby the damaged crystalline lens is replaced by a synthetic lens. Although cataract surgery is highly effective, a relatively common complication named posterior capsular opacification (PCO) leads to secondary [...] Read more.

Cataract is the leading cause of blindness worldwide. It can be treated by surgery, whereby the damaged crystalline lens is replaced by a synthetic lens. Although cataract surgery is highly effective, a relatively common complication named posterior capsular opacification (PCO) leads to secondary loss of vision. PCO is caused by abnormal proliferation and migration of residual lens epithelial cells (LECs) that were not removed during the surgery, which results in interruption to the passage of light. Despite technical improvements to the surgery, this complication has not been eradicated. Efforts are being made to identify drugs that can be applied post-surgery, to inhibit PCO development. Towards the goal of identifying such drugs, we used zebrafish embryos homozygous for a mutation in plod3 that develop a lens phenotype with characteristics of PCO. Using both biased and unbiased approaches, we identified small molecules that can block the lens phenotype of the mutants. Our findings confirm the relevance of zebrafish plod3 mutants’ lens phenotype as a model for lens epithelium-derived cataract and add to our understanding of the molecular mechanisms that contribute to the development of this pathology. This understanding should help in the development of strategies for PCO prevention. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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24 pages, 6647 KiB

Open AccessArticle

Eye Lens Organoids Made Simple: Characterization of a New Three-Dimensional Organoid Model for Lens Development and Pathology

by Matthieu Duot, Roselyne Viel, Justine Viet, Catherine Le Goff-Gaillard, Luc Paillard, Salil A. Lachke, Carole Gautier-Courteille and David Reboutier

Cells 2023, 12(20), 2478; https://doi.org/10.3390/cells12202478 - 18 Oct 2023

Cited by 1 | Viewed by 1467

Abstract

Cataract, the opacification of the lens, is the leading cause of blindness worldwide. Although effective, cataract surgery is costly and can lead to complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and drug screening. Here, [...] Read more.

Cataract, the opacification of the lens, is the leading cause of blindness worldwide. Although effective, cataract surgery is costly and can lead to complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined three-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization and biology. These organoids can be rapidly produced in large amounts. High-throughput RNA sequencing (RNA-seq) on specific organoid regions isolated via laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display a spatiotemporal expression of key lens genes, e.g., Jag1, Pax6, Prox1, Hsf4 and Cryab. Further, these lens organoids are amenable to the induction of opacities. Finally, the knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1, induces opacities in these organoids, indicating their use in rapidly screening for genes that are functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataracts. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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19 pages, 4054 KiB

Open AccessArticle

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix

by Miguel Jarrin, Alexia A. Kalligeraki, Alice Uwineza, Chris S. Cawood, Adrian P. Brown, Edward N. Ward, Khoa Le, Stefanie Freitag-Pohl, Ehmke Pohl, Bence Kiss, Antal Tapodi and Roy A. Quinlan

Cells 2023, 12(12), 1580; https://doi.org/10.3390/cells12121580 - 07 Jun 2023

Cited by 1 | Viewed by 1287

Abstract

Background: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434–452 were α-helical [...] Read more.

Background: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434–452 were α-helical and amphipathic. Methods and Results: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434–467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. Conclusions: These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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20 pages, 20166 KiB

Open AccessArticle

Spatiotemporal Localisation of Heparan Sulphate Proteoglycans throughout Mouse Lens Morphogenesis

by Tayler F. L. Wishart and Frank J. Lovicu

Cells 2023, 12(10), 1364; https://doi.org/10.3390/cells12101364 - 11 May 2023

Cited by 1 | Viewed by 1528

Abstract

Heparan sulphate proteoglycans (HSPGs) consist of a core protein decorated with sulphated HS-glycosaminoglycan (GAG) chains. These negatively charged HS-GAG chains rely on the activity of PAPSS synthesising enzymes for their sulfation, which allows them to bind to and regulate the activity of many [...] Read more.

Heparan sulphate proteoglycans (HSPGs) consist of a core protein decorated with sulphated HS-glycosaminoglycan (GAG) chains. These negatively charged HS-GAG chains rely on the activity of PAPSS synthesising enzymes for their sulfation, which allows them to bind to and regulate the activity of many positively charged HS-binding proteins. HSPGs are found on the surfaces of cells and in the pericellular matrix, where they interact with various components of the cell microenvironment, including growth factors. By binding to and regulating ocular morphogens and growth factors, HSPGs are positioned to orchestrate growth factor-mediated signalling events that are essential for lens epithelial cell proliferation, migration, and lens fibre differentiation. Previous studies have shown that HS sulfation is essential for lens development. Moreover, each of the full-time HSPGs, differentiated by thirteen different core proteins, are differentially localised in a cell-type specific manner with regional differences in the postnatal rat lens. Here, the same thirteen HSPG-associated GAGs and core proteins as well as PAPSS2, are shown to be differentially regulated throughout murine lens development in a spatiotemporal manner. These findings suggest that HS-GAG sulfation is essential for growth factor-induced cellular processes during embryogenesis, and the unique and divergent localisation of different lens HSPG core proteins indicates that different HSPGs likely play specialized roles during lens induction and morphogenesis. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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21 pages, 3972 KiB

Open AccessArticle

High-Throughput Transcriptomics of Celf1 Conditional Knockout Lens Identifies Downstream Networks Linked to Cataract Pathology

by Archana D. Siddam, Matthieu Duot, Sarah Y. Coomson, Deepti Anand, Sandeep Aryal, Bailey A. T. Weatherbee, Yann Audic, Luc Paillard and Salil A. Lachke

Cells 2023, 12(7), 1070; https://doi.org/10.3390/cells12071070 - 01 Apr 2023

Cited by 5 | Viewed by 1775

Abstract

Defects in the development of the ocular lens can cause congenital cataracts. To understand the various etiologies of congenital cataracts, it is important to characterize the genes linked to this developmental defect and to define their downstream pathways that are relevant to lens [...] Read more.

Defects in the development of the ocular lens can cause congenital cataracts. To understand the various etiologies of congenital cataracts, it is important to characterize the genes linked to this developmental defect and to define their downstream pathways that are relevant to lens biology and pathology. Deficiency or alteration of several RNA-binding proteins, including the conserved RBP Celf1 (CUGBP Elav-like family member 1), has been described to cause lens defects and early onset cataracts in animal models and/or humans. Celf1 is involved in various aspects of post-transcriptional gene expression control, including regulation of mRNA stability/decay, alternative splicing and translation. Celf1 germline knockout mice and lens conditional knockout (Celf1^cKO) mice develop fully penetrant cataracts in early postnatal stages. To define the genome-level changes in RNA transcripts that result from Celf1 deficiency, we performed high-throughput RNA-sequencing of Celf1^cKO mouse lenses at postnatal day (P) 0. Celf1^cKO lenses exhibit 987 differentially expressed genes (DEGs) at cut-offs of >1.0 log2 counts per million (CPM), ≥±0.58 log2 fold-change and <0.05 false discovery rate (FDR). Of these, 327 RNAs were reduced while 660 were elevated in Celf1^cKO lenses. The DEGs were subjected to various downstream analyses including iSyTE lens enriched-expression, presence in Cat-map, and gene ontology (GO) and representation of regulatory pathways. Further, a comparative analysis was done with previously generated microarray datasets on Celf1^cKO lenses P0 and P6. Together, these analyses validated and prioritized several key genes mis-expressed in Celf1^cKO lenses that are relevant to lens biology, including known cataract-linked genes (e.g., Cryab, Cryba2, Cryba4, Crybb1, Crybb2, Cryga, Crygb, Crygc, Crygd, Cryge, Crygf, Dnase2b, Bfsp1, Gja3, Pxdn, Sparc, Tdrd7, etc.) as well as novel candidates (e.g., Ell2 and Prdm16). Together, these data have defined the alterations in lens transcriptome caused by Celf1 deficiency, in turn uncovering downstream genes and pathways (e.g., structural constituents of eye lenses, lens fiber cell differentiation, etc.) associated with lens development and early-onset cataracts. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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20 pages, 3173 KiB

Open AccessArticle

FGF-2 Differentially Regulates Lens Epithelial Cell Behaviour during TGF-β-Induced EMT

by Mary Flokis and Frank J. Lovicu

Cells 2023, 12(6), 827; https://doi.org/10.3390/cells12060827 - 07 Mar 2023

Cited by 5 | Viewed by 1710

Abstract

Fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β) can regulate and/or dysregulate lens epithelial cell (LEC) behaviour, including proliferation, fibre differentiation, and epithelial–mesenchymal transition (EMT). Earlier studies have investigated the crosstalk between FGF and TGF-β in dictating lens cell fate, that appears [...] Read more.

Fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-β) can regulate and/or dysregulate lens epithelial cell (LEC) behaviour, including proliferation, fibre differentiation, and epithelial–mesenchymal transition (EMT). Earlier studies have investigated the crosstalk between FGF and TGF-β in dictating lens cell fate, that appears to be dose dependent. Here, we tested the hypothesis that a fibre-differentiating dose of FGF differentially regulates the behaviour of lens epithelial cells undergoing TGF-β-induced EMT. Postnatal 21-day-old rat lens epithelial explants were treated with a fibre-differentiating dose of FGF-2 (200 ng/mL) and/or TGF-β2 (50 pg/mL) over a 7-day culture period. We compared central LECs (CLECs) and peripheral LECs (PLECs) using immunolabelling for changes in markers for EMT (α-SMA), lens fibre differentiation (β-crystallin), epithelial cell adhesion (β-catenin), and the cytoskeleton (alpha-tropomyosin), as well as Smad2/3- and MAPK/ERK1/2-signalling. Lens epithelial explants cotreated with FGF-2 and TGF-β2 exhibited a differential response, with CLECs undergoing EMT while PLECs favoured more of a lens fibre differentiation response, compared to the TGF-β-only-treated explants where all cells in the explants underwent EMT. The CLECs cotreated with FGF and TGF-β immunolabelled for α-SMA, with minimal β-crystallin, whereas the PLECs demonstrated strong β-crystallin reactivity and little α-SMA. Interestingly, compared to the TGF-β-only-treated explants, α-SMA was significantly decreased in the CLECs cotreated with FGF/TGF-β. Smad-dependent and independent signalling was increased in the FGF-2/TGF-β2 co-treated CLECs, that had a heightened number of cells with nuclear localisation of Smad2/3 compared to the PLECs, that in contrast had more pronounced ERK1/2-signalling over Smad2/3 activation. The current study has confirmed that FGF-2 is influential in differentially regulating the behaviour of LECs during TGF-β-induced EMT, leading to a heterogenous cell population, typical of that observed in the development of post-surgical, posterior capsular opacification (PCO). This highlights the cooperative relationship between FGF and TGF-β leading to lens pathology, providing a different perspective when considering preventative measures for controlling PCO. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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28 pages, 4532 KiB

Open AccessArticle

Lens Epithelial Explants Treated with Vitreous Humor Undergo Alterations in Chromatin Landscape with Concurrent Activation of Genes Associated with Fiber Cell Differentiation and Innate Immune Response

by Anil Upreti, Stephanie L. Padula, Jared A. Tangeman, Brad D. Wagner, Michael J. O’Connell, Tycho J. Jaquish, Raye K. Palko, Courtney J. Mantz, Deepti Anand, Frank J. Lovicu, Salil A. Lachke and Michael L. Robinson

Cells 2023, 12(3), 501; https://doi.org/10.3390/cells12030501 - 03 Feb 2023

Cited by 4 | Viewed by 2143

Abstract

Lens epithelial explants are comprised of lens epithelial cells cultured in vitro on their native basement membrane, the lens capsule. Biologists have used lens epithelial explants to study many different cellular processes including lens fiber cell differentiation. In these studies, fiber differentiation is [...] Read more.

Lens epithelial explants are comprised of lens epithelial cells cultured in vitro on their native basement membrane, the lens capsule. Biologists have used lens epithelial explants to study many different cellular processes including lens fiber cell differentiation. In these studies, fiber differentiation is typically measured by cellular elongation and the expression of a few proteins characteristically expressed by lens fiber cells in situ. Chromatin and RNA was collected from lens epithelial explants cultured in either un-supplemented media or media containing 50% bovine vitreous humor for one or five days. Chromatin for ATAC-sequencing and RNA for RNA-sequencing was prepared from explants to assess regions of accessible chromatin and to quantitatively measure gene expression, respectively. Vitreous humor increased chromatin accessibility in promoter regions of genes associated with fiber differentiation and, surprisingly, an immune response, and this was associated with increased transcript levels for these genes. In contrast, vitreous had little effect on the accessibility of the genes highly expressed in the lens epithelium despite dramatic reductions in their mRNA transcripts. An unbiased analysis of differentially accessible regions revealed an enrichment of cis-regulatory motifs for RUNX, SOX and TEAD transcription factors that may drive differential gene expression in response to vitreous. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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13 pages, 3154 KiB

Open AccessArticle

Imaging Cataract-Specific Peptides in Human Lenses

by Kevin L. Schey, Zhen Wang, Kristie L. Rose and David M. G. Anderson

Cells 2022, 11(24), 4042; https://doi.org/10.3390/cells11244042 - 14 Dec 2022

Cited by 2 | Viewed by 1421

Abstract

Age-related protein truncation is a common process in long-lived proteins such as proteins found in the ocular lens. Major truncation products have been reported for soluble and membrane proteins of the lens, including small peptides that can accelerate protein aggregation. However, the spatial [...] Read more.

Age-related protein truncation is a common process in long-lived proteins such as proteins found in the ocular lens. Major truncation products have been reported for soluble and membrane proteins of the lens, including small peptides that can accelerate protein aggregation. However, the spatial localization of age-related protein fragments in the lens has received only limited study. Imaging mass spectrometry (IMS) is an ideal tool for examining the spatial localization of protein products in tissues. In this study we used IMS to determine the spatial localization of small crystallin fragments in aged and cataractous lenses. Consistent with previous reports, the pro-aggregatory αA-crystallin 66–80 peptide as well as αA-crystallin 67–80 and γS-crystallin 167–178 were detected in normal lenses, but found to be increased in nuclear cataract regions. In addition, a series of γS-crystallin C-terminal peptides were observed to be mainly localized to cataractous regions and barely detected in transparent lenses. Other peptides, including abundant αA3-crystallin peptides were present in both normal and cataract lenses. The functional properties of these crystallin peptides remain unstudied; however, their cataract-specific localization suggests further studies are warranted. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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16 pages, 3644 KiB

Open AccessArticle

PI3K Isoform-Specific Regulation of Leader and Follower Cell Function for Collective Migration and Proliferation in Response to Injury

by Morgan D. Basta, A. Sue Menko and Janice L. Walker

Cells 2022, 11(21), 3515; https://doi.org/10.3390/cells11213515 - 07 Nov 2022

Cited by 1 | Viewed by 1676

Abstract

To ensure proper wound healing it is important to elucidate the signaling cues that coordinate leader and follower cell behavior to promote collective migration and proliferation for wound healing in response to injury. Using an ex vivo post-cataract surgery wound healing model we [...] Read more.

To ensure proper wound healing it is important to elucidate the signaling cues that coordinate leader and follower cell behavior to promote collective migration and proliferation for wound healing in response to injury. Using an ex vivo post-cataract surgery wound healing model we investigated the role of class I phosphatidylinositol-3-kinase (PI3K) isoforms in this process. Our findings revealed a specific role for p110α signaling independent of Akt for promoting the collective migration and proliferation of the epithelium for wound closure. In addition, we found an important role for p110α signaling in orchestrating proper polarized cytoskeletal organization within both leader and wounded epithelial follower cells to coordinate their function for wound healing. p110α was necessary to signal the formation and persistence of vimentin rich-lamellipodia extensions by leader cells and the reorganization of actomyosin into stress fibers along the basal domains of the wounded lens epithelial follower cells for movement. Together, our study reveals a critical role for p110α in the collective migration of an epithelium in response to wounding. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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28 pages, 4781 KiB

Open AccessArticle

The Immediate Early Response of Lens Epithelial Cells to Lens Injury

by Samuel G. Novo, Adam P. Faranda, Mahbubul H. Shihan, Yan Wang, Ananya Garg and Melinda K. Duncan

Cells 2022, 11(21), 3456; https://doi.org/10.3390/cells11213456 - 01 Nov 2022

Cited by 4 | Viewed by 1997

Abstract

Cataracts are treated by lens fiber cell removal followed by intraocular lens (IOL) implantation into the lens capsule. While effective, this procedure leaves behind numerous lens epithelial cells (LECs) which undergo a wound healing response that frequently leads to posterior capsular opacification (PCO). [...] Read more.

Cataracts are treated by lens fiber cell removal followed by intraocular lens (IOL) implantation into the lens capsule. While effective, this procedure leaves behind numerous lens epithelial cells (LECs) which undergo a wound healing response that frequently leads to posterior capsular opacification (PCO). In order to elucidate the acute response of LECs to lens fiber cell removal which models cataract surgery (post cataract surgery, PCS), RNA-seq was conducted on LECs derived from wild type mice at 0 and 6 h PCS. This analysis found that LECs upregulate the expression of numerous proinflammatory cytokines and profibrotic regulators by 6 h PCS suggesting rapid priming of pathways leading to inflammation and fibrosis PCS. LECs also highly upregulate the expression of numerous immediate early transcription factors (IETFs) by 6 h PCS and immunolocalization found elevated levels of these proteins by 3 h PCS, and this was preceded by the phosphorylation of ERK1/2 in injured LECs. Egr1 and FosB were among the highest expressed of these factors and qRT-PCR revealed that they also upregulate in explanted mouse lens epithelia suggesting potential roles in the LEC injury response. Analysis of lenses lacking either Egr1 or FosB revealed that both genes may regulate a portion of the acute LEC injury response, although neither gene was essential for expression of either proinflammatory or fibrotic markers at later times PCS suggesting that IETFs may work in concert to mediate the LEC injury response following cataract surgery. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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12 pages, 1947 KiB

Open AccessArticle

Mapping the Universe of Eph Receptor and Ephrin Ligand Transcripts in Epithelial and Fiber Cells of the Eye Lens

by Michael P. Vu and Catherine Cheng

Cells 2022, 11(20), 3291; https://doi.org/10.3390/cells11203291 - 19 Oct 2022

Cited by 1 | Viewed by 1360

Abstract

The eye lens is a transparent, ellipsoid organ in the anterior chamber of the eye that is required for fine focusing of light onto the retina to transmit a clear image. Cataracts, defined as any opacity in the lens, remains the leading cause [...] Read more.

The eye lens is a transparent, ellipsoid organ in the anterior chamber of the eye that is required for fine focusing of light onto the retina to transmit a clear image. Cataracts, defined as any opacity in the lens, remains the leading cause of blindness in the world. Recent studies in humans and mice indicate that Eph–ephrin bidirectional signaling is important for maintaining lens transparency. Specifically, mutations and polymorphisms in the EphA2 receptor and the ephrin-A5 ligand have been linked to congenital and age-related cataracts. It is unclear what other variants of Ephs and ephrins are expressed in the lens or whether there is preferential expression in epithelial vs. fiber cells. We performed a detailed analysis of Eph receptor and ephrin ligand mRNA transcripts in whole mouse lenses, epithelial cell fractions, and fiber cell fractions using a new RNA isolation method. We compared control samples with EphA2 knockout (KO) and ephrin-A5 KO samples. Our results revealed the presence of transcripts for 12 out of 14 Eph receptors and 8 out of 8 ephrin ligands in various fractions of lens cells. Using specific primer sets, RT-PCR, and sequencing, we verified the variant of each gene that is expressed, and we found two epithelial-cell-specific genes. Surprisingly, we also identified one Eph receptor variant that is expressed in KO lens fibers but is absent from control lens fibers. We also identified one low expression ephrin variant that is only expressed in ephrin-A5 control samples. These results indicate that the lens expresses almost all Ephs and ephrins, and there may be many receptor–ligand pairs that play a role in lens homeostasis. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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13 pages, 1146 KiB

Open AccessArticle

Levels and Modifications of Both Lens Fiber Cell Connexins Are Affected in Connexin Mutant Mice

by Oscar Jara, Peter J. Minogue, Viviana M. Berthoud and Eric C. Beyer

Cells 2022, 11(18), 2786; https://doi.org/10.3390/cells11182786 - 07 Sep 2022

Cited by 1 | Viewed by 1125

Abstract

In the lens, cell homeostasis and transparency are supported by intercellular communication facilitated by the channels formed of connexin46 (Cx46) and connexin50 (Cx50). Mutations of these connexins are linked to inherited cataracts. We studied the levels and the variations in electrophoretic mobilities of [...] Read more.

In the lens, cell homeostasis and transparency are supported by intercellular communication facilitated by the channels formed of connexin46 (Cx46) and connexin50 (Cx50). Mutations of these connexins are linked to inherited cataracts. We studied the levels and the variations in electrophoretic mobilities of the immunoreactive Cx46 and Cx50 bands between 1 and 21 days after birth in the lenses of wild-type mice and homozygous animals from two different mouse models of connexin-linked cataracts (Cx46fs380 and Cx50D47A). In Cx50D47A mice, the expression of the mutant Cx50 reduced the normal phosphorylation of the co-expressed wild-type Cx46. In both models, levels of the mutant connexin and the co-expressed wild-type connexin decayed more rapidly than in wild-type mice but with different time courses. In the Cx46fs380 mice, modeling suggested that Cx50 degradation could be explained by the mixing of mutant Cx46 with wild-type Cx50. However, in Cx50D47A mice, similar modeling suggested that mixing alone could not explain the decrease in Cx46 levels. These data highlight the complex influences between two connexin proteins expressed in the same cell, some of which occur through direct mixing, while others occur indirectly, as in Cx50D47A mice, where the expression of the mutant connexin causes endoplasmic reticulum stress and impaired differentiation. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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13 pages, 2832 KiB

Open AccessArticle

Double Deletion of PI3K and PTEN Modifies Lens Postnatal Growth and Homeostasis

by Caterina Sellitto, Leping Li and Thomas W. White

Cells 2022, 11(17), 2708; https://doi.org/10.3390/cells11172708 - 30 Aug 2022

Viewed by 1282

Abstract

We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of [...] Read more.

We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of phosphorylated Akt and equatorial epithelial cell proliferation, and resulted in smaller transparent lenses in adult mice. The deletion of PTEN increased levels of phosphorylated Akt, altered lens sodium transport, and caused lens rupture and cataract. Here, we have generated conditional p110α/PTEN double-knockout mice, and evaluated epithelial cell proliferation and lens homeostasis. The double deletion of p110α and PTEN rescued the defect in lens size seen after the single knockout of p110α, but accelerated the lens rupture phenotype seen in PTEN single-knockout mice. Levels of phosphorylated Akt in double-knockout lenses were significantly higher than in wild-type lenses, but not as elevated as those reported for PTEN single-knockout lenses. These results showed that the double deletion of the p110α catalytic subunit of PI3K and its opposing phosphatase, PTEN, exacerbated the rupture defect seen in the single PTEN knockout and alleviated the growth defect observed in the single p110α knockout. Thus, the integrity of the PI3K signaling pathway was absolutely essential for proper lens homeostasis, but not for lens growth. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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19 pages, 10162 KiB

Open AccessArticle

Aged Lens Epithelial Cells Suppress Proliferation and Epithelial–Mesenchymal Transition-Relevance for Posterior Capsule Opacification

by Zongbo Wei, Pasley Gordon, Caili Hao, Jingru Huangfu, Emily Fan, Xiang Zhang, Hong Yan and Xingjun Fan

Cells 2022, 11(13), 2001; https://doi.org/10.3390/cells11132001 - 22 Jun 2022

Cited by 7 | Viewed by 2294

Abstract

Posterior capsule opacification (PCO) is a frequent complication after cataract surgery, and advanced PCO requires YAG laser (Nd: YAG) capsulotomy, which often gives rise to more complications. Lens epithelial cell (LEC) proliferation and transformation (i.e., epithelial–mesenchymal transition (EMT)) are two critical elements in [...] Read more.

Posterior capsule opacification (PCO) is a frequent complication after cataract surgery, and advanced PCO requires YAG laser (Nd: YAG) capsulotomy, which often gives rise to more complications. Lens epithelial cell (LEC) proliferation and transformation (i.e., epithelial–mesenchymal transition (EMT)) are two critical elements in PCO initiation and progression pathogenesis. While PCO marginally impacts aged cataract surgery patients, PCO incidences are exceptionally high in infants and children undergoing cataract surgery. The gene expression of lens epithelial cell aging and its role in the discrepancy of PCO prevalence between young and older people have not been fully studied. Here, we conducted a comprehensive differentially expressed gene (DEG) analysis of a cell aging model by comparing the early and late passage FHL124 lens epithelial cells (LECs). In vitro, TGFβ2, cell treatment, and in vivo mouse cataract surgical models were used to validate our findings. We found that aged LECs decelerated rates of cell proliferation accompanied by dysregulation of cellular immune response and cell stress response. Surprisingly, we found that LECs systematically downregulated epithelial–mesenchymal transition (EMT)-promoting genes. The protein expression of several EMT hallmark genes, e.g., fibronectin, αSMA, and cadherin 11, were gradually decreased during LECs aging. We then confirmed these findings in vitro and found that aged LECs markedly alleviated TGFβ2-mediated EMT. Importantly, we explicitly confirmed the in vitro findings from the in vivo mouse cataract surgery studies. We propose that both the high proliferation rate and EMT-enriched young LECs phenotypic characteristics contribute to unusually high PCO incidence in infants and children. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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18 pages, 4521 KiB

Open AccessReview

Myo/Nog Cells: The Jekylls and Hydes of the Lens

by Jacquelyn Gerhart and Mindy George-Weinstein

Cells 2023, 12(13), 1725; https://doi.org/10.3390/cells12131725 - 27 Jun 2023

Viewed by 1708

Abstract

Herein, we review a unique and versatile lineage composed of Myo/Nog cells that may be beneficial or detrimental depending on their environment and nature of the pathological stimuli they are exposed to. While we will focus on the lens, related Myo/Nog cell behaviors [...] Read more.

Herein, we review a unique and versatile lineage composed of Myo/Nog cells that may be beneficial or detrimental depending on their environment and nature of the pathological stimuli they are exposed to. While we will focus on the lens, related Myo/Nog cell behaviors and functions in other tissues are integrated into the narrative of our research that spans over three decades, examines multiple species and progresses from early stages of embryonic development to aging adults. Myo/Nog cells were discovered in the embryonic epiblast by their co-expression of the skeletal muscle-specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin and brain-specific angiogenesis inhibitor 1. They were tracked from the epiblast into the developing lens, revealing heterogeneity of cell types within this structure. Depletion of Myo/Nog cells in the epiblast results in eye malformations arising from the absence of Noggin. In the adult lens, Myo/Nog cells are the source of myofibroblasts whose contractions produce wrinkles in the capsule. Eliminating this population within the rabbit lens during cataract surgery reduces posterior capsule opacification to below clinically significant levels. Parallels are drawn between the therapeutic potential of targeting Myo/Nog cells to prevent fibrotic disease in the lens and other ocular tissues. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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21 pages, 9690 KiB

Open AccessReview

Autophagy Requirements for Eye Lens Differentiation and Transparency

by Lisa Brennan, M. Joseph Costello, J. Fielding Hejtmancik, A. Sue Menko, S. Amer Riazuddin, Alan Shiels and Marc Kantorow

Cells 2023, 12(3), 475; https://doi.org/10.3390/cells12030475 - 01 Feb 2023

Cited by 5 | Viewed by 2213

Abstract

Recent evidence points to autophagy as an essential cellular requirement for achieving the mature structure, homeostasis, and transparency of the lens. Collective evidence from multiple laboratories using chick, mouse, primate, and human model systems provides evidence that classic autophagy structures, ranging from double-membrane [...] Read more.

Recent evidence points to autophagy as an essential cellular requirement for achieving the mature structure, homeostasis, and transparency of the lens. Collective evidence from multiple laboratories using chick, mouse, primate, and human model systems provides evidence that classic autophagy structures, ranging from double-membrane autophagosomes to single-membrane autolysosomes, are found throughout the lens in both undifferentiated lens epithelial cells and maturing lens fiber cells. Recently, key autophagy signaling pathways have been identified to initiate critical steps in the lens differentiation program, including the elimination of organelles to form the core lens organelle-free zone. Other recent studies using ex vivo lens culture demonstrate that the low oxygen environment of the lens drives HIF1a-induced autophagy via upregulation of essential mitophagy components to direct the specific elimination of the mitochondria, endoplasmic reticulum, and Golgi apparatus during lens fiber cell differentiation. Pioneering studies on the structural requirements for the elimination of nuclei during lens differentiation reveal the presence of an entirely novel structure associated with degrading lens nuclei termed the nuclear excisosome. Considerable evidence also indicates that autophagy is a requirement for lens homeostasis, differentiation, and transparency, since the mutation of key autophagy proteins results in human cataract formation. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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18 pages, 1591 KiB

Open AccessReview

Role of Decorin in the Lens and Ocular Diseases

by Eri Kubo, Shinsuke Shibata, Teppei Shibata, Hiroshi Sasaki and Dhirendra P. Singh

Cells 2023, 12(1), 74; https://doi.org/10.3390/cells12010074 - 24 Dec 2022

Cited by 2 | Viewed by 2407

Abstract

Decorin is an archetypal member of the small leucine-rich proteoglycan gene family and is involved in various biological functions and many signaling networks, interacting with extra-cellular matrix (ECM) components, growth factors, and receptor tyrosine kinases. Decorin also modulates the growth factors, cell proliferation, [...] Read more.

Decorin is an archetypal member of the small leucine-rich proteoglycan gene family and is involved in various biological functions and many signaling networks, interacting with extra-cellular matrix (ECM) components, growth factors, and receptor tyrosine kinases. Decorin also modulates the growth factors, cell proliferation, migration, and angiogenesis. It has been reported to be involved in many ischemic and fibrotic eye diseases, such as congenital stromal dystrophy of the cornea, anterior subcapsular fibrosis of the lens, proliferative vitreoretinopathy, et al. Furthermore, recent evidence supports its role in secondary posterior capsule opacification (PCO) after cataract surgery. The expression of decorin mRNA in lens epithelial cells in vitro was found to decrease upon transforming growth factor (TGF)-β-2 addition and increase upon fibroblast growth factor (FGF)-2 addition. Wound healing of the injured lens in mice transgenic for lens-specific human decorin was promoted by inhibiting myofibroblastic changes. Decorin may be associated with epithelial–mesenchymal transition and PCO development in the lens. Gene therapy and decorin administration have the potential to serve as excellent therapeutic approaches for modifying impaired wound healing, PCO, and other eye diseases related to fibrosis and angiogenesis. In this review, we present findings regarding the roles of decorin in the lens and ocular diseases. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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42 pages, 1992 KiB

Open AccessFeature PaperEditor’s ChoiceReview

Generation of Lens Progenitor Cells and Lentoid Bodies from Pluripotent Stem Cells: Novel Tools for Human Lens Development and Ocular Disease Etiology

by Aleš Cvekl and Michael John Camerino

Cells 2022, 11(21), 3516; https://doi.org/10.3390/cells11213516 - 06 Nov 2022

Cited by 5 | Viewed by 2999

Abstract

In vitro differentiation of human pluripotent stem cells (hPSCs) into specialized tissues and organs represents a powerful approach to gain insight into those cellular and molecular mechanisms regulating human development. Although normal embryonic eye development is a complex process, generation of ocular organoids [...] Read more.

In vitro differentiation of human pluripotent stem cells (hPSCs) into specialized tissues and organs represents a powerful approach to gain insight into those cellular and molecular mechanisms regulating human development. Although normal embryonic eye development is a complex process, generation of ocular organoids and specific ocular tissues from pluripotent stem cells has provided invaluable insights into the formation of lineage-committed progenitor cell populations, signal transduction pathways, and self-organization principles. This review provides a comprehensive summary of recent advances in generation of adenohypophyseal, olfactory, and lens placodes, lens progenitor cells and three-dimensional (3D) primitive lenses, “lentoid bodies”, and “micro-lenses”. These cells are produced alone or “community-grown” with other ocular tissues. Lentoid bodies/micro-lenses generated from human patients carrying mutations in crystallin genes demonstrate proof-of-principle that these cells are suitable for mechanistic studies of cataractogenesis. Taken together, current and emerging advanced in vitro differentiation methods pave the road to understand molecular mechanisms of cataract formation caused by the entire spectrum of mutations in DNA-binding regulatory genes, such as PAX6, SOX2, FOXE3, MAF, PITX3, and HSF4, individual crystallins, and other genes such as BFSP1, BFSP2, EPHA2, GJA3, GJA8, LIM2, MIP, and TDRD7 represented in human cataract patients. Full article

(This article belongs to the Special Issue New Advances in Lens Biology and Pathology)

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