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Gupta N, Somayajulu M, Gurdziel K, LoGrasso G, Aziz H, Rosati R, McClellan S, Pitchaikannu A, Santra M, Shukkur MFA, Stemmer P, Hazlett LD, Xu S. The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell type-specific target genes. Sci Rep 2024; 14:7676. [PMID: 38561433 PMCID: PMC10985120 DOI: 10.1038/s41598-024-58403-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2023] [Accepted: 03/28/2024] [Indexed: 04/04/2024] Open
Abstract
The conserved miR-183/96/182 cluster (miR-183C) is expressed in both corneal resident myeloid cells (CRMCs) and sensory nerves (CSN) and modulates corneal immune/inflammatory responses. To uncover cell type-specific roles of miR-183C in CRMC and CSN and their contributions to corneal physiology, myeloid-specific miR-183C conditional knockout (MS-CKO), and sensory nerve-specific CKO (SNS-CKO) mice were produced and characterized in comparison to the conventional miR-183C KO. Immunofluorescence and confocal microscopy of flatmount corneas, corneal sensitivity, and tear volume assays were performed in young adult naïve mice; 3' RNA sequencing (Seq) and proteomics in the trigeminal ganglion (TG), cornea and CRMCs. Our results showed that, similar to conventional KO mice, the numbers of CRMCs were increased in both MS-CKO and SNS-CKO vs age- and sex-matched WT control littermates, suggesting intrinsic and extrinsic regulations of miR-183C on CRMCs. The number of CRMCs was increased in male vs female MS-CKO mice, suggesting sex-dependent regulation of miR-183C on CRMCs. In the miR-183C KO and SNS-CKO, but not the MS-CKO mice, CSN density was decreased in the epithelial layer of the cornea, but not the stromal layer. Functionally, corneal sensitivity and basal tear volume were reduced in the KO and SNS-CKO, but not the MS-CKO mice. Tear volume in males is consistently higher than female WT mice. Bioinformatic analyses of the transcriptomes revealed a series of cell-type specific target genes of miR-183C in TG sensory neurons and CRMCs. Our data elucidate that miR-183C imposes intrinsic and extrinsic regulation on the establishment and function of CSN and CRMCs by cell-specific target genes. miR-183C modulates corneal sensitivity and tear production through its regulation of corneal sensory innervation.
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Affiliation(s)
- Naman Gupta
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 E Canfield Street, Detroit, MI, 48201, USA
| | - Mallika Somayajulu
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 E Canfield Street, Detroit, MI, 48201, USA
| | | | - Giovanni LoGrasso
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 E Canfield Street, Detroit, MI, 48201, USA
| | - Haidy Aziz
- School of Biological Sciences, Wayne State University, Detroit, MI, USA
| | - Rita Rosati
- Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA
| | - Sharon McClellan
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 E Canfield Street, Detroit, MI, 48201, USA
| | - Ahalya Pitchaikannu
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 E Canfield Street, Detroit, MI, 48201, USA
| | - Manoranjan Santra
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 E Canfield Street, Detroit, MI, 48201, USA
| | - Muhammed Farooq Abdul Shukkur
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 E Canfield Street, Detroit, MI, 48201, USA
| | - Paul Stemmer
- Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA
| | - Linda D Hazlett
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 E Canfield Street, Detroit, MI, 48201, USA
| | - Shunbin Xu
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, 540 E Canfield Street, Detroit, MI, 48201, USA.
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Hopkinson A, Notara M, Cursiefen C, Sidney LE. Increased Anti-Inflammatory Therapeutic Potential and Progenitor Marker Expression of Corneal Mesenchymal Stem Cells Cultured in an Optimized Propagation Medium. Cell Transplant 2024; 33:9636897241241992. [PMID: 38602231 PMCID: PMC11010753 DOI: 10.1177/09636897241241992] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Revised: 02/19/2024] [Accepted: 03/05/2024] [Indexed: 04/12/2024] Open
Abstract
There is a huge unmet need for new treatment modalities for ocular surface inflammatory disorders (OSIDs) such as dry eye disease and meibomian gland dysfunction. Mesenchymal stem cell therapies may hold the answer due to their potent immunomodulatory properties, low immunogenicity, and ability to modulate both the innate and adaptive immune response. MSC-like cells that can be isolated from the corneal stroma (C-MSCs) offer a potential new treatment strategy; however, an optimized culture medium needs to be developed to produce the ideal phenotype for use in a cell therapy to treat OSIDs. The effects of in vitro expansion of human C-MSC in a medium of M199 containing fetal bovine serum (FBS) was compared to a stem cell medium (SCM) containing knockout serum replacement (KSR) with basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (LIF), investigating viability, protein, and gene expression. Isolating populations expressing CD34 or using siRNA knockdown of CD34 were investigated. Finally, the potential of C-MSC as a cell therapy was assessed using co-culture with an in vitro corneal epithelial cell injury model and the angiogenic effects of C-MSC conditioned medium were evaluated with blood and lymph endothelial cells. Both media supported proliferation of C-MSC, with SCM increasing expression of CD34, ABCG2, PAX6, NANOG, REX1, SOX2, and THY1, supported by increased associated protein expression. Isolating cell populations expressing CD34 protein made little difference to gene expression, however, knockdown of the CD34 gene led to decreased expression of progenitor genes. C-MSC increased viability of injured corneal epithelial cells whilst decreasing levels of cytotoxicity and interleukins-6 and -8. No pro-angiogenic effect of C-MSC was seen. Culture medium can significantly influence C-MSC phenotype and culture in SCM produced a cell phenotype more suitable for further consideration as an anti-inflammatory cell therapy. C-MSC show considerable potential for development as therapies for OSIDs, acting through anti-inflammatory action.
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Affiliation(s)
- Andrew Hopkinson
- Academic Ophthalmology, Mental Health and Clinical Neurosciences, University of Nottingham, Nottingham, UK
| | - Maria Notara
- Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Koln, Germany
| | - Claus Cursiefen
- Department of Ophthalmology, Faculty of Medicine and University Hospital Cologne, University of Cologne, Koln, Germany
| | - Laura E. Sidney
- Academic Ophthalmology, Mental Health and Clinical Neurosciences, University of Nottingham, Nottingham, UK
- Regenerating and Modelling Tissues, Translational Medical Sciences, School of Medicine, University of Nottingham, Nottingham, UK
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Lu X, Chen Z, Lu J, Watsky MA. Effects of 1,25-Vitamin D3 and 24,25-Vitamin D3 on Corneal Nerve Regeneration in Diabetic Mice. Biomolecules 2023; 13:1754. [PMID: 38136625 PMCID: PMC10742127 DOI: 10.3390/biom13121754] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2023] [Revised: 11/30/2023] [Accepted: 12/02/2023] [Indexed: 12/24/2023] Open
Abstract
Corneal nerve homeostasis is essential for the functional integrity of the ocular surface. Vitamin D deficiency (VDD) and vitamin D receptor knockout (VDR KO) have been found to reduce corneal nerve density in diabetic mice. This is the first study to comprehensively examine the influence of vitamin D on nerve regeneration following corneal epithelial injury in diabetic mice. Corneal nerve regeneration was significantly retarded by diabetes, VDR KO, and VDD, and it was accelerated following topical 1,25 Vit D and 24,25 Vit D administration. Furthermore, topical 1,25 Vit D and 24,25 Vit D increased nerve growth factor, glial cell line-derived neurotropic factor, and neurotropin-3 protein expression, and it increased secretion of GDNF protein from human corneal epithelial cells. CD45+ cells and macrophage numbers were significantly decreased, and vitamin D increased CD45+ cell and macrophage recruitment in these wounded diabetic mouse corneas. The accelerated nerve regeneration observed in these corneas following topical 1,25 Vit D and 24,25 Vit D administration may be related to the vitamin D-stimulated expression, secretion of neurotrophic factors, and recruitment of immune cells.
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Affiliation(s)
- Xiaowen Lu
- Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, 1120 15th Street, CB-2901, Augusta, GA 30912, USA
| | | | | | - Mitchell A. Watsky
- Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta University, 1120 15th Street, CB-2901, Augusta, GA 30912, USA
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Li W, Gurdziel K, Pitchaikannu A, Gupta N, Hazlett LD, Xu S. The miR-183/96/182 cluster is a checkpoint for resident immune cells and shapes the cellular landscape of the cornea. Ocul Surf 2023; 30:17-41. [PMID: 37536656 PMCID: PMC10834862 DOI: 10.1016/j.jtos.2023.07.012] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Revised: 07/28/2023] [Accepted: 07/31/2023] [Indexed: 08/05/2023]
Abstract
PURPOSE The conserved miR-183/96/182 cluster (miR-183C) regulates both corneal sensory innervation and corneal resident immune cells (CRICs). This study is to uncover its role in CRICs and in shaping the corneal cellular landscape at a single-cell (sc) level. METHODS Corneas of naïve, young adult [2 and 6 months old (mo)], female miR-183C knockout (KO) mice and wild-type (WT) littermates were harvested and dissociated into single cells. Dead cells were removed using a Dead Cell Removal kit. CD45+ CRICs were enriched by Magnetic Activated Cell Sorting (MACS). scRNA libraries were constructed and sequenced followed by comprehensive bioinformatic analyses. RESULTS The composition of major cell types of the cornea stays relatively stable in WT mice from 2 to 6 mo, however the compositions of subtypes of corneal cells shift with age. Inactivation of miR-183C disrupts the stability of the major cell-type composition and age-related transcriptomic shifts of subtypes of corneal cells. The diversity of CRICs is enhanced with age. Naïve mouse cornea contains previously-unrecognized resident fibrocytes and neutrophils. Resident macrophages (ResMφ) adopt cornea-specific function by expressing abundant extracellular matrix (ECM) and ECM organization-related genes. Naïve cornea is endowed with partially-differentiated proliferative ResMφ and contains microglia-like Mφ. Resident lymphocytes, including innate lymphoid cells (ILCs), NKT and γδT cells, are the major source of innate IL-17a. miR-183C limits the diversity and polarity of ResMφ. CONCLUSION miR-183C serves as a checkpoint for CRICs and imposes a global regulation of the cellular landscape of the cornea.
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Affiliation(s)
- Weifeng Li
- Predoctoral Training Program in Human Genetics, McKusick-Nathans Institute of Genetic Medicine, Department of Genetic Medicine, USA; Wilmer Eye Institute, School of Medicine, The Johns Hopkins University, Baltimore, MD, USA
| | | | - Ahalya Pitchaikannu
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, Detroit, MI, USA
| | - Naman Gupta
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, Detroit, MI, USA
| | - Linda D Hazlett
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, Detroit, MI, USA
| | - Shunbin Xu
- Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, Detroit, MI, USA.
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Lu X, Chen Z, Lu J, Watsky M. Effects of Topical 1,25 and 24,25 Vitamin D on Diabetic, Vitamin D Deficient and Vitamin D Receptor Knockout Mouse Corneal Wound Healing. Biomolecules 2023; 13:1065. [PMID: 37509101 PMCID: PMC10377579 DOI: 10.3390/biom13071065] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2023] [Revised: 06/26/2023] [Accepted: 06/28/2023] [Indexed: 07/30/2023] Open
Abstract
Delayed or prolonged corneal wound healing and non-healing corneas put patients at risk for ocular surface infections and subsequent stromal opacification, resulting in discomfort or visual loss. It is important to enhance corneal wound healing efficiency and quality. Vitamin D (Vit D) is both a hormone and a vitamin, and its insufficiency has been linked to immune disorders and diabetes. For this study, wound healing and recruitment of CD45+ cells into the wound area of normoglycemic and diabetic mice were examined following corneal epithelial debridement and treatment with 1,25-dihyroxyvitamin D (1,25 Vit D) or 24,25-dihydroxyvitamin D (24,25 Vit D). Treatment with topical 1,25-dihyroxyvitamin D (1,25 Vit D) resulted in significantly increased corneal wound healing rates of normoglycemic, diabetic and diabetic Vit D deficient mice. Furthermore, 24,25-dihydroxyvitamin D (24,25 Vit D) significantly increased corneal wound healing of diabetic Vit D deficient and Vit D receptor knockout (VDR KO) mice. In addition, CD45+ cell numbers were reduced in diabetic and VDR KO mouse corneas compared to normoglycemic mice, and 24,25 Vit D increased the recruitment of CD45+ cells to diabetic mouse corneas after epithelial debridement. CD45+ cells were found to infiltrate into the corneal basal epithelial layer after corneal epithelial debridement. Our data indicate that topical Vit D promotes corneal wound healing and further supports previous work that the Vit D corneal wound healing effect is not totally VDR-dependent.
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Affiliation(s)
| | | | | | - Mitchell Watsky
- Department of Cellular Biology and Anatomy, Medical College of Georgia at Augusta University, Augusta, GA 30912, USA
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Gulka SMD, Gowen B, Litke AM, Delaney KR, Chow RL. Laser-induced microinjury of the corneal basal epithelium and imaging of resident macrophage responses in a live, whole-eye preparation. Front Immunol 2023; 14:1050594. [PMID: 36814930 PMCID: PMC9939765 DOI: 10.3389/fimmu.2023.1050594] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Accepted: 01/23/2023] [Indexed: 02/09/2023] Open
Abstract
The corneal epithelium is continuously subjected to external stimuli that results in varying degrees of cellular damage. The use of live-cell imaging approaches has facilitated understanding of the cellular and molecular mechanisms underlying the corneal epithelial wound healing process. Here, we describe a live, ex vivo, whole-eye approach using laser scanning confocal microscopy to simultaneously induce and visualize short-term cellular responses following microdamage to the corneal epithelium. Live-cell imaging of corneal cell layers was enabled using the lipophilic fluorescent dyes, SGC5 or FM4-64, which, when injected into the anterior chamber of enucleated eyes, readily penetrated and labelled cell membranes. Necrotic microdamage to a defined region (30 μm x 30 μm) through the central plane of the corneal basal epithelium was induced by continuously scanning for at least one minute using high laser power and was dependent on the presence of lipophilic fluorescent dye. This whole-mount live-cell imaging and microdamage approach was used to examine the behavior of Cx3cr1:GFP-expressing resident corneal stromal macrophages (RCSMs). In undamaged corneas, RCSMs remained stationary, but exhibited a constant extension and retraction of short (~5 μm) semicircular, pseudopodia-like processes reminiscent of what has previously been reported in corneal dendritic cells. Within minutes of microdamage, nearby anterior RCSMs became highly polarized and extended projections towards the damaged region. The extension of the processes plateaued after about 30 minutes and remained stable over the course of 2-3 hours of imaging. Retrospective immunolabeling showed that these responding RCSMs were MHC class II+. This study adds to existing knowledge of immune cell behavior in response to corneal damage and introduces a simple corneal epithelial microdamage and wound healing paradigm.
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Affiliation(s)
- Sebastian M. D. Gulka
- Department of Biology, University of Victoria, Victoria, BC, Canada
- University of Illinois College of Medicine, Chicago, IL, United States
| | - Brent Gowen
- Department of Biology, University of Victoria, Victoria, BC, Canada
| | | | - Kerry R. Delaney
- Department of Biology, University of Victoria, Victoria, BC, Canada
| | - Robert L. Chow
- Department of Biology, University of Victoria, Victoria, BC, Canada
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Tavakkoli F, Eleiwa TK, Elhusseiny AM, Damala M, Rai AK, Cheraqpour K, Ansari MH, Doroudian M, H Keshel S, Soleimani M, Djalilian AR, Sangwan VS, Singh V. Corneal stem cells niche and homeostasis impacts in regenerative medicine; concise review. Eur J Ophthalmol 2023:11206721221150065. [PMID: 36604831 DOI: 10.1177/11206721221150065] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
The limbal stem cells niche (LSCN) is an optimal microenvironment that provides the limbal epithelial stem cells (LESCs) and strictly regulates their proliferation and differentiation. Disturbing the LSCN homeostasis can lead to limbal stem cell dysfunction (LSCD) and subsequent ocular surface aberrations, such as corneal stromal inflammation, persistent epithelial defects, corneal neovascularisation, lymphangiogenesis, corneal opacification, and conjunctivalization. As ocular surface disorders are considered the second main cause of blindness, it becomes crucial to explore different therapeutic strategies for restoring the functions of the LSCN. A major limitation of corneal transplantation is the current shortage of donor tissue to meet the requirements worldwide. In this context, it becomes mandatory to find an alternative regenerative medicine, such as using cultured limbal epithelial/stromal stem cells, inducing the production of corneal like cells by using other sources of stem cells, and using tissue engineering methods aiming to produce the three-dimensional (3D) printed cornea. Limbal epithelial stem cells have been considered the magic potion for eye treatment. Epithelial and stromal stem cells in the limbal niche hold the responsibility of replenishing the corneal epithelium. These stem cells are being used for transplantation to maintain corneal epithelial integrity and ultimately sustain optimal vision. In this review, we summarised the characteristics of the LSCN and their current and future roles in restoring corneal homeostasis in eyes with LSCD.
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Affiliation(s)
- Fatemeh Tavakkoli
- Department of Community Health, College of Health Technology, Cihan University, Erbil, Iraq.,SSR Stem Cell Biology Laboratory, Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad, India.,Centre for Genetic Disorders, Banaras Hindu University, Varanasi, India
| | - Taher K Eleiwa
- Department of Ophthalmology, Benha University, Benha, Egypt
| | - Abdelrahman M Elhusseiny
- Department of Ophthalmology, Harvey and Bernice Jones Eye Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA
| | - Mukesh Damala
- SSR Stem Cell Biology Laboratory, Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad, India.,School of Life Sciences, University of Hyderabad, Hyderabad, India
| | - Amit K Rai
- Centre for Genetic Disorders, Banaras Hindu University, Varanasi, India
| | - Kasra Cheraqpour
- Translational Eye Research Center, Farabi Eye Hospital, 48439Tehran University of Medical Sciences, Tehran, Iran
| | - Mohammad H Ansari
- Ophthalmic Research Center, Department of Ophthalmology, Labbafinejad Medical Center, 556492Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Mohammad Doroudian
- Department of Cell and Molecular Biology, Faculty of Biological Sciences, 145440Kharazmi University, Tehran, Iran
| | - Saeed H Keshel
- Department of Tissue Engineering and Applied Cell Sciences, 556492Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Mohammad Soleimani
- Department of Ophthalmology, 159636Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, IL, USA
| | - Ali R Djalilian
- Department of Ophthalmology, 159636Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, IL, USA
| | | | - Vivek Singh
- SSR Stem Cell Biology Laboratory, Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, Hyderabad, India
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Filiberti A, Gmyrek GB, Berube AN, Carr DJJ. Osteopontin contributes to virus resistance associated with type I IFN expression, activation of downstream ifn-inducible effector genes, and CCR2 +CD115 +CD206 + macrophage infiltration following ocular HSV-1 infection of mice. Front Immunol 2023; 13:1028341. [PMID: 36685562 PMCID: PMC9846535 DOI: 10.3389/fimmu.2022.1028341] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Accepted: 12/05/2022] [Indexed: 01/06/2023] Open
Abstract
Ocular pathology is often associated with acute herpes simplex virus (HSV)-1 infection of the cornea in mice. The present study was undertaken to determine the role of early T lymphocyte activation 1 protein or osteopontin (OPN) in corneal inflammation and host resistance to ocular HSV-1 infection. C57BL/6 wild type (WT) and osteopontin deficient (OPN KO) mice infected in the cornea with HSV-1 were evaluated for susceptibility to infection and cornea pathology. OPN KO mice were found to possess significantly more infectious virus in the cornea at day 3 and day 7 post infection compared to infected WT mice. Coupled with these findings, HSV-1-infected OPN KO mouse corneas were found to express less interferon (IFN)-α1, double-stranded RNA-dependent protein kinase, and RNase L compared to infected WT animals early post infection that likely contributed to decreased resistance. Notably, OPN KO mice displayed significantly less corneal opacity and neovascularization compared to WT mice that paralleled a decrease in expression of vascular endothelial growth factor (VEGF) A within 12 hr post infection. The change in corneal pathology of the OPN KO mice aligned with a decrease in total leukocyte infiltration into the cornea and specifically, in neutrophils at day 3 post infection and in macrophage subpopulations including CCR2+CD115+CD206+ and CD115+CD183+CD206+ -expressing cells. The infiltration of CD4+ and CD8+ T cells into the cornea was unaltered comparing infected WT to OPN KO mice. Likewise, there was no difference in the total number of HSV-1-specific CD4+ or CD8+ T cells found in the draining lymph node with both sets functionally competent in response to virus antigen comparing WT to OPN KO mice. Collectively, these results demonstrate OPN deficiency directly influences the host innate immune response to ocular HSV-1 infection reducing some aspects of inflammation but at a cost with an increase in local HSV-1 replication.
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Affiliation(s)
- Adrian Filiberti
- Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
| | - Grzegorz B. Gmyrek
- Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
| | - Amanda N. Berube
- Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
| | - Daniel J. J. Carr
- Department of Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
- Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States
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Neuroimmune crosstalk in the cornea: The role of immune cells in corneal nerve maintenance during homeostasis and inflammation. Prog Retin Eye Res 2022; 91:101105. [PMID: 35868985 DOI: 10.1016/j.preteyeres.2022.101105] [Citation(s) in RCA: 43] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Revised: 07/01/2022] [Accepted: 07/04/2022] [Indexed: 12/29/2022]
Abstract
In the cornea, resident immune cells are in close proximity to sensory nerves, consistent with their important roles in the maintenance of nerves in both homeostasis and inflammation. Using in vivo confocal microscopy in humans, and ex vivo immunostaining and fluorescent reporter mice to visualize corneal sensory nerves and immune cells, remarkable progress has been made to advance our understanding of the physical and functional interactions between corneal nerves and immune cells. In this review, we summarize and discuss recent studies relating to corneal immune cells and sensory nerves, and their interactions in health and disease. In particular, we consider how disrupted corneal nerve axons can induce immune cell activity, including in dendritic cells, macrophages and other infiltrating cells, directly and/or indirectly by releasing neuropeptides such as substance P and calcitonin gene-related peptide. We summarize growing evidence that the role of corneal intraepithelial immune cells is likely different in corneal wound healing versus other inflammatory-dominated conditions. The role of different types of macrophages is also discussed, including how stromal macrophages with anti-inflammatory phenotypes communicate with corneal nerves to provide neuroprotection, while macrophages with pro-inflammatory phenotypes, along with other infiltrating cells including neutrophils and CD4+ T cells, can be inhibitory to corneal re-innervation. Finally, this review considers the bidirectional interactions between corneal immune cells and corneal nerves, and how leveraging this interaction could represent a potential therapeutic approach for corneal neuropathy.
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Kitazawa K, Sotozono C, Kinoshita S. Current Advancements in Corneal Cell-Based Therapy. Asia Pac J Ophthalmol (Phila) 2022; 11:335-345. [PMID: 36041148 DOI: 10.1097/apo.0000000000000530] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2021] [Accepted: 03/10/2022] [Indexed: 12/13/2022] Open
Abstract
Corneal epithelial stem cells (CEpSCs) mostly reside at the limbal area and are responsible for tissue homeostasis throughout life. Once complete CEpSC deficiency occurs, regenerative medicine cell-based therapy using CEpSCs or their alternatives can provide successful clinical outcomes. Due to an improved understanding of CEpSCs and mucosal epithelial stem cells, major advancements have been made over the past few decades in in vivo and ex vivo cell-based ocular surface reconstruction therapies for the treatment of severe ocular surface diseases. New therapeutic concepts and clinical strategies are emerging for the treatment of corneal endothelial dysfunction. For example, unlike corneal epithelial cells, in vivo corneal endothelial cells (CECs) stop proliferating and are arrested in the G1 phase of the cell cycle due to cell-to-cell contact inhibition and exposure to a high concentration of transforming growth factor-beta in the aqueous humor. Thus, the production of CECs with good functionality in culture has consistently been difficult. To solve this problem, Rho-associated protein kinase inhibition has taken center stage, as it not only makes the production of human CECs in culture closely mimic the functional characteristics of in vivo healthy CECs possible but also helps sustain those biological properties. Thus, cultured human CEC injection therapy is now moving to the forefront for the treatment of corneal endothelial failure. Herein, we summarize key historical discoveries in corneal cell-based regenerative medicine and illustrate the concept of corneal cell therapy for the treatment of refractory corneal epithelial and endothelial diseases.
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Affiliation(s)
- Koji Kitazawa
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Chie Sotozono
- Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
| | - Shigeru Kinoshita
- Department of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
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11
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Rajaiya J, Saha A, Zhou X, Chodosh J. Human Adenovirus Species D Interactions with Corneal Stromal Cells. Viruses 2021; 13:2505. [PMID: 34960773 PMCID: PMC8709199 DOI: 10.3390/v13122505] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2021] [Revised: 12/11/2021] [Accepted: 12/13/2021] [Indexed: 11/17/2022] Open
Abstract
Notable among the many communicable agents known to infect the human cornea is the human adenovirus, with less than ten adenoviruses having corneal tropism out of more than 100 known types. The syndrome of epidemic keratoconjunctivitis (EKC), caused principally by human adenovirus, presents acutely with epithelial keratitis, and later with stromal keratitis that can be chronic and recurrent. In this review, we discuss the current state of knowledge regarding the molecular biology of adenovirus infection of corneal stromal cells, among which the fibroblast-like keratocyte is the most predominant, in order to elucidate basic pathophysiologic mechanisms of stromal keratitis in the human patient with EKC.
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Affiliation(s)
- Jaya Rajaiya
- Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA; (A.S.); (X.Z.)
| | | | | | - James Chodosh
- Massachusetts Eye and Ear, Harvard Medical School, Boston, MA 02114, USA; (A.S.); (X.Z.)
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12
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Thomson BR, Liu P, Onay T, Du J, Tompson SW, Misener S, Purohit RR, Young TL, Jin J, Quaggin SE. Cellular crosstalk regulates the aqueous humor outflow pathway and provides new targets for glaucoma therapies. Nat Commun 2021; 12:6072. [PMID: 34663817 PMCID: PMC8523664 DOI: 10.1038/s41467-021-26346-0] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2020] [Accepted: 09/30/2021] [Indexed: 11/09/2022] Open
Abstract
Primary congenital glaucoma (PCG) is a severe disease characterized by developmental defects in the trabecular meshwork (TM) and Schlemm's canal (SC), comprising the conventional aqueous humor outflow pathway of the eye. Recently, heterozygous loss of function variants in TEK and ANGPT1 or compound variants in TEK/SVEP1 were identified in children with PCG. Moreover, common variants in ANGPT1and SVEP1 have been identified as risk alleles for primary open angle glaucoma (POAG) in GWAS studies. Here, we show tissue-specific deletion of Angpt1 or Svep1 from the TM causes PCG in mice with severe defects in the adjacent SC. Single-cell transcriptomic analysis of normal and glaucomatous Angpt1 deficient eyes allowed us to identify distinct TM and SC cell populations and discover additional TM-SC signaling pathways. Furthermore, confirming the importance of angiopoietin signaling in SC, delivery of a recombinant ANGPT1-mimetic promotes developmental SC expansion in healthy and Angpt1 deficient eyes, blunts intraocular pressure (IOP) elevation and RGC loss in a mouse model of PCG and lowers IOP in healthy adult mice. Our data highlight the central role of ANGPT1-TEK signaling and TM-SC crosstalk in IOP homeostasis and provide new candidates for SC-targeted glaucoma therapy.
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Affiliation(s)
- Benjamin R Thomson
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
- The Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA
| | - Pan Liu
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
- The Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA
| | - Tuncer Onay
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
| | - Jing Du
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
- The Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA
| | - Stuart W Tompson
- Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA
| | - Sol Misener
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
- The Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA
| | - Raj R Purohit
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
- The Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA
| | - Terri L Young
- Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA
| | - Jing Jin
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
- The Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA
| | - Susan E Quaggin
- Division of Nephrology and Hypertension, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
- The Feinberg Cardiovascular and Renal Research Institute, Chicago, IL, USA.
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Abstract
This review describes a group of diseases known as the transmissible spongiform encephalopathies (TSEs), which affect animals and humans. Examination of affected brain tissue suggests that these diseases are caused by the acquisition and deposition of prion protein (PrP). Creutzfeldt-Jakob disease (CJD) is the most important form of TSE in humans with at least four different varieties of the disease. Variant CJD (vCJD), a new form of the disease found in the UK, has several features that differ from the classical forms including early age of onset, longer duration of disease, psychiatric presentation (for example, depression) and extensive florid plaque development in the brain. About 10 per cent of patients with CJD exhibit visual symptoms at disease presentation and approximately 50 per cent during the course of the disease. The most commonly reported visual symptoms include diplopia, supranuclear palsies, complex visual disturbances, homonymous visual field defects, hallucinations and cortical blindness. Saccadic and smooth pursuit movements appear to be more rarely affected. The agent causing vCJD accumulates in lymphoid tissue such as the spleen and tonsils. The cornea has lymphoid tissue in the form of corneal dendritic cells that are important in the regulation of the immune response in the anterior segment of the eye. The presence of these cells in the cornea has raised the possibility of transmission between patients via optical devices that contact the eye. Although such transmission is theoretically possible it remains highly improbable.
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14
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Wilson SE, Sampaio LP, Shiju TM, Carlos de Oliveira R. Fibroblastic and bone marrow-derived cellularity in the corneal stroma. Exp Eye Res 2020; 202:108303. [PMID: 33068626 DOI: 10.1016/j.exer.2020.108303] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2020] [Revised: 09/18/2020] [Accepted: 10/12/2020] [Indexed: 11/16/2022]
Abstract
The unwounded, normal corneal stroma is a relatively simple, avascular tissue populated with quiescent keratocytes, along with corneal nerves and a few resident dendritic and monocyte/macrophage cells. In the past, the resting keratocytes were thought of as a homogenous cellular population, but recent work has shown local variations in vimentin and nestin expression, and responsiveness to transforming growth factor (TGF)-β1. Studies have also supported there being "stromal stem cells" in localized areas. After corneal wounding, depending on the site and severity of injury, profound changes in stromal cellularity occur. Anterior or posterior injuries to the epithelium or endothelium, respectively, trigger apoptosis of adjacent keratocytes. Many contiguous keratocytes transition to keratocan-negative corneal fibroblasts that are proliferative and produce limited amounts of disorganized extracellular matrix components. Simultaneously, large numbers of bone marrow-derived cells, including monocytes, neutrophils, fibrocytes and lymphocytes, invade the stroma from the limbal blood vessels. Ongoing adequate levels of TGFβ1, TGFβ2 and platelet-derived growth factor (PDGF) from epithelium, tears, endothelium and aqueous humor that penetrate defective or absent epithelial barrier function (EBF) and epithelial basement membrane (EBM) and/or Descemet's basement membrane (DBM) drive corneal fibroblasts and fibrocytes to differentiate into alpha-smooth muscle actin (SMA)-positive myofibroblasts. If the EBF, EBM and/or DBM are repaired or replaced in a timely manner, typically measured in weeks, then corneal fibroblast and fibrocyte progeny, deprived of requisite levels of TGFβ1 and TGFβ2, undergo apoptosis or revert to their precursor cell-types. If the EBF, EBM and/or DBM are not repaired or replaced, stromal levels of TGFβ1 and TGFβ2 remain elevated, and mature myofibroblasts are generated from corneal fibroblasts and fibrocyte precursors that produce prodigious amounts of disordered extracellular matrix materials associated with scarring fibrosis. This fibrotic stromal matrix persists, at least until the EBF, EBM and/or DBM are regenerated or replaced, and keratocytes remove and reorganize the affected stromal matrix.
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Affiliation(s)
- Steven E Wilson
- Cole Eye Institute, I-32, Cleveland Clinic, 9500, Euclid Ave, Cleveland, OH, United States.
| | - Lycia Pedral Sampaio
- Cole Eye Institute, I-32, Cleveland Clinic, 9500, Euclid Ave, Cleveland, OH, United States
| | - Thomas Michael Shiju
- Cole Eye Institute, I-32, Cleveland Clinic, 9500, Euclid Ave, Cleveland, OH, United States
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15
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Jamali A, Kenyon B, Ortiz G, Abou-Slaybi A, Sendra VG, Harris DL, Hamrah P. Plasmacytoid dendritic cells in the eye. Prog Retin Eye Res 2020; 80:100877. [PMID: 32717378 DOI: 10.1016/j.preteyeres.2020.100877] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2020] [Revised: 05/28/2020] [Accepted: 06/05/2020] [Indexed: 02/07/2023]
Abstract
Plasmacytoid dendritic cells (pDCs) are a unique subpopulation of immune cells, distinct from classical dendritic cells. pDCs are generated in the bone marrow and following development, they typically home to secondary lymphoid tissues. While peripheral tissues are generally devoid of pDCs during steady state, few tissues, including the lung, kidney, vagina, and in particular ocular tissues harbor resident pDCs. pDCs were originally appreciated for their potential to produce large quantities of type I interferons in viral immunity. Subsequent studies have now unraveled their pivotal role in mediating immune responses, in particular in the induction of tolerance. In this review, we summarize our current knowledge on pDCs in ocular tissues in both mice and humans, in particular in the cornea, limbus, conjunctiva, choroid, retina, and lacrimal gland. Further, we will review our current understanding on the significance of pDCs in ameliorating inflammatory responses during herpes simplex virus keratitis, sterile inflammation, and corneal transplantation. Moreover, we describe their novel and pivotal neuroprotective role, their key function in preserving corneal angiogenic privilege, as well as their potential application as a cell-based therapy for ocular diseases.
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Affiliation(s)
- Arsia Jamali
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA
| | - Brendan Kenyon
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Program in Neuroscience, Graduate School of Biomedical Sciences, Tufts University, Boston, MA, USA
| | - Gustavo Ortiz
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA
| | - Abdo Abou-Slaybi
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Program in Immunology, Graduate School of Biomedical Sciences, Tufts University, Boston, MA, USA
| | - Victor G Sendra
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA
| | - Deshea L Harris
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA
| | - Pedram Hamrah
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Boston, MA, USA; Program in Neuroscience, Graduate School of Biomedical Sciences, Tufts University, Boston, MA, USA; Program in Immunology, Graduate School of Biomedical Sciences, Tufts University, Boston, MA, USA; Cornea Service, Tufts New England Eye Center, Boston, MA, USA.
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16
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Corneal Stem Cells as a Source of Regenerative Cell-Based Therapy. Stem Cells Int 2020; 2020:8813447. [PMID: 32765614 PMCID: PMC7388005 DOI: 10.1155/2020/8813447] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Revised: 07/03/2020] [Accepted: 07/10/2020] [Indexed: 12/15/2022] Open
Abstract
In the past few years, intensive research has focused on corneal stem cells as an unlimited source for cell-based therapy in regenerative ophthalmology. Today, it is known that the cornea has at least two types of stem cells: limbal epithelial stem cells (LESCs) and corneal stromal stem cells (CSSCs). LESCs are used for regeneration of corneal surface, while CSSCs are used for regeneration of corneal stroma. Until now, various approaches and methods for isolation of LESCs and CSSCs and their successful transplantation have been described and tested in several preclinical studies and clinical trials. This review describes in detail phenotypic characteristics of LESCs and CSSCs and discusses their therapeutic potential in corneal regeneration. Since efficient and safe corneal stem cell-based therapy is still a challenging issue that requires continuous cooperation between researchers, clinicians, and patients, this review addresses the important limitations and suggests possible strategies for improvement of corneal stem cell-based therapy.
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17
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Yam GHF, Riau AK, Funderburgh ML, Mehta JS, Jhanji V. Keratocyte biology. Exp Eye Res 2020; 196:108062. [PMID: 32442558 DOI: 10.1016/j.exer.2020.108062] [Citation(s) in RCA: 44] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2020] [Revised: 05/01/2020] [Accepted: 05/04/2020] [Indexed: 12/12/2022]
Abstract
The study of corneal stromal keratocytes is motivated by its strong association with corneal health and visual function. They play a dominant role in the maintenance of corneal homeostasis and transparency through the production of collagens, proteoglycans and corneal crystallins. Trauma-induced apoptosis of keratocytes and replacement by fibroblasts and myofibroblasts disrupt the stromal matrix organization, resulting in corneal haze formation and vision loss. It is, therefore, important to understand the biology and behaviours of keratocytes and the associated stromal cell types (like fibroblasts, myofibroblasts, stromal stem cells) in wound healing, corneal pathologies (including keratoconus, keratitis, endothelial disorders) as well as different ophthalmic situations (such as collagen crosslinking/photodynamic treatment, keratoplasty and refractive surgery, and topical medications). The recent development of ex vivo propagation of keratocytes and stromal stem cells, and their translational applications, either via stromal injection or incorporated in bioscaffold, have been shown to restore the corneal transparency and regenerate native stromal tissue in animal models of corneal haze and other disorders.
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Affiliation(s)
- Gary H F Yam
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA.
| | - Andri K Riau
- Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore
| | | | - Jodhbir S Mehta
- Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore
| | - Vishal Jhanji
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA
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18
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Cellular Factor XIII, a Transglutaminase in Human Corneal Keratocytes. Int J Mol Sci 2019; 20:ijms20235963. [PMID: 31783511 PMCID: PMC6928837 DOI: 10.3390/ijms20235963] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2019] [Revised: 11/20/2019] [Accepted: 11/25/2019] [Indexed: 02/06/2023] Open
Abstract
Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.
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19
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Yam GHF, Seah X, Yusoff NZBM, Setiawan M, Wahlig S, Htoon HM, Peh GSL, Kocaba V, Mehta JS. Characterization of Human Transition Zone Reveals a Putative Progenitor-Enriched Niche of Corneal Endothelium. Cells 2019; 8:cells8101244. [PMID: 31614883 PMCID: PMC6829622 DOI: 10.3390/cells8101244] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2019] [Revised: 10/09/2019] [Accepted: 10/10/2019] [Indexed: 12/12/2022] Open
Abstract
: The corneal endothelium regulates corneal hydration to maintain the transparency of cornea. Lacking regenerative capacity, corneal endothelial cell loss due to aging and diseases can lead to corneal edema and vision loss. There is limited information on the existence of corneal endothelial progenitors. We conducted ultrastructural examinations and expression analyses on the human transition zone (TZ) at the posterior limbus of corneal periphery, to elucidate if the TZ harbored progenitor-like cells, and to reveal their niche characteristics. Within the narrow TZ (~190 μm width), the inner TZ-adjacent to the peripheral endothelium (PE)-contained cells expressing stem/progenitor markers (Sox2, Lgr5, CD34, Pitx2, telomerase). They were located on the inner TZ surface and in its underlying stroma. Lgr5 positive cells projected as multicellular clusters into the PE. Under transmission electron microscopy and serial block face-scanning electron microscopy and three-dimensional (3D) reconstruction, the terminal margin of Descemet's membrane was inserted beneath the TZ surface, with the distance akin to the inner TZ breadth. Porcine TZ cells were isolated and proliferated into a confluent monolayer and differentiated to cells expressing corneal endothelial markers (ZO1, Na+K+ATPase) on cell surface. In conclusion, we have identified a novel inner TZ containing progenitor-like cells, which could serve the regenerative potential for corneal endothelium.
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Affiliation(s)
- Gary Hin-Fai Yam
- Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
- Eye-Academic Clinical Program, Duke-National University of Singapore (NUS) Graduate Medical School, Singapore 169857, Singapore.
| | - Xinyi Seah
- Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
| | | | - Melina Setiawan
- Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
| | - Stephen Wahlig
- Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
- Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27705, USA.
| | - Hla Myint Htoon
- Eye-Academic Clinical Program, Duke-National University of Singapore (NUS) Graduate Medical School, Singapore 169857, Singapore.
- Data Science Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
| | - Gary S L Peh
- Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
- Eye-Academic Clinical Program, Duke-National University of Singapore (NUS) Graduate Medical School, Singapore 169857, Singapore.
| | - Viridiana Kocaba
- Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
- Department of Ophthalmology, Claude Bernard Lyon 1 Université, 69622 Villeurbanne, France.
| | - Jodhbir S Mehta
- Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
- Eye-Academic Clinical Program, Duke-National University of Singapore (NUS) Graduate Medical School, Singapore 169857, Singapore.
- Singapore National Eye Centre, Singapore, Singapore 168751, Singapore.
- School of Material Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore.
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20
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Seyed-Razavi Y, Lopez MJ, Mantopoulos D, Zheng L, Massberg S, Sendra VG, Harris DL, Hamrah P. Kinetics of corneal leukocytes by intravital multiphoton microscopy. FASEB J 2019; 33:2199-2211. [PMID: 30226811 PMCID: PMC6338630 DOI: 10.1096/fj.201800684rr] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2018] [Accepted: 08/27/2018] [Indexed: 12/13/2022]
Abstract
Corneal immune privilege is integral in maintaining the clear avascular window to the foreign world. The presence of distinct populations of corneal leukocytes (CLs) in the normal cornea has been firmly established. However, their precise function and kinetics remain, as of yet, unclear. Through intravital multiphoton microscopy (IV-MPM), allowing the means to accumulate critical spatial and temporal cellular information, we provide details for long-term investigation of CL morphology and kinetics under steady state and following inflammation. Significant alterations in size and morphology of corneal CD11c+ dendritic cells (DCs) were noted following acute sterile inflammation, including cell volume (4364.4 ± 489.6 vs. 1787.6 ± 111.0 μm3, P < 0.001) and sphericity (0.82 ± 0.01 vs. 0.42 ± 0.02, P < 0.001) compared with steady state. Furthermore, IV-MPM analyses revealed alterations in both the CD11c+ DC and major histocompatibility complex class II (MHC)-II+ mature antigen-presenting cell population kinetics during inflammation, including track displacement length (CD11c: 16.57 ± 1.41 vs. 4.64 ± 0.56 μm, P < 0.001; MHC-II: 9.03 ± 0.37 vs. 4.09 ± 0.39, P < 0.001) and velocity (CD11c: 1.91 ± 0.07 μm/min vs. 1.73 ± 0.1302 μm/min; MHC-II: 2.97 ± 0.07 vs. 1.62 ± 0.08, P < 0.001) compared with steady state. Our results reveal in vivo evidence of sessile CL populations exhibiting dendritic morphology under steady state and increased velocity of spherical leukocytes following inflammation. IV-MPM represents a powerful tool to study leukocytes in corneal diseases in context.-Seyed-Razavi, Y., Lopez, M. J., Mantopoulos, D., Zheng, L., Massberg, S., Sendra, V. G., Harris, D. L., Hamrah, P. Kinetics of corneal leukocytes by intravital multiphoton microscopy.
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Affiliation(s)
- Yashar Seyed-Razavi
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Schepens Eye Research Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
| | - Maria J. Lopez
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Schepens Eye Research Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
| | - Dimosthenis Mantopoulos
- Schepens Eye Research Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
- Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA
| | - Lixin Zheng
- Schepens Eye Research Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
- Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA
| | - Steffen Massberg
- Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA
- Department of Cardiology, Ludwig Maximilians Universität, Munich, Germany
| | - Victor G. Sendra
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Schepens Eye Research Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
| | - Deshea L. Harris
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Schepens Eye Research Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
| | - Pedram Hamrah
- Center for Translational Ocular Immunology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Department of Ophthalmology, Tufts Medical Center, Tufts University School of Medicine, Tufts University, Boston, Massachusetts, USA
- Schepens Eye Research Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
- Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA
- Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA
- Cornea Service, Tufts New England Eye Center, Boston, Massachusetts, USA
- Cornea Service, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
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21
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Lassance L, Marino GK, Medeiros CS, Thangavadivel S, Wilson SE. Fibrocyte migration, differentiation and apoptosis during the corneal wound healing response to injury. Exp Eye Res 2018; 170:177-187. [PMID: 29481786 DOI: 10.1016/j.exer.2018.02.018] [Citation(s) in RCA: 71] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2017] [Revised: 01/30/2018] [Accepted: 02/23/2018] [Indexed: 01/03/2023]
Abstract
The aim of this study was to determine whether bone marrow-derived fibrocytes migrate into the cornea after stromal scar-producing injury and differentiate into alpha-smooth muscle actin (αSMA) + myofibroblasts. Chimeric mice expressing green fluorescent protein (GFP) bone marrow cells had fibrosis (haze)-generating irregular phototherapeutic keratectomy (PTK). Multiplex immunohistochemistry (IHC) for GFP and fibrocyte markers (CD34, CD45, and vimentin) was used to detect fibrocyte infiltration into the corneal stroma and the development of GFP+ αSMA+ myofibroblasts. IHC for activated caspase-3, GFP and CD45 was used to detect fibrocyte and other hematopoietic cells undergoing apoptosis. Moderate haze developed in PTK-treated mouse corneas at 14 days after surgery and worsened, and persisted, at 21 days after surgery. GFP+ CD34+ CD45+ fibrocytes, likely in addition to other CD34+ and/or CD45+ hematopoietic and stem/progenitor cells, infiltrated the cornea and were present in the stroma in high numbers by one day after PTK. The fibrocytes and other bone marrow-derived cells progressively decreased at four days and seven days after surgery. At four days after PTK, 5% of the GFP+ cells expressed activated caspase-3. At 14 days after PTK, more than 50% of GFP+ CD45+ cells were also αSMA+ myofibroblasts. At 21 days after PTK, few GFP+ αSMA+ cells persisted in the stroma and more than 95% of those remaining expressed activated caspase-3, indicating they were undergoing apoptosis. GFP+ CD45+ SMA+ cells that developed from 4 to 21 days after irregular PTK were likely developed from fibrocytes. After irregular PTK in the strain of C57BL/6-C57/BL/6-Tg(UBC-GFP)30Scha/J chimeric mice, however, more than 95% of fibrocytes and other hematopoietic cells underwent apoptosis prior to the development of mature αSMA+ myofibroblasts. Most GFP+ CD45+ αSMA+ myofibroblasts that did develop subsequently underwent apoptosis-likely due to epithelial basement membrane regeneration and deprivation of epithelium-derived TGFβ requisite for myofibroblast survival.
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Affiliation(s)
- Luciana Lassance
- Cole Eye Institute, Cleveland Clinic, Cleveland, OH, United States
| | | | - Carla S Medeiros
- Cole Eye Institute, Cleveland Clinic, Cleveland, OH, United States; University of Sao Paulo, Sao Paulo, Brazil
| | | | - Steven E Wilson
- Cole Eye Institute, Cleveland Clinic, Cleveland, OH, United States.
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22
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Lolli F, Pallotti F, Rossi A, Fortuna MC, Caro G, Lenzi A, Sansone A, Lombardo F. Androgenetic alopecia: a review. Endocrine 2017; 57:9-17. [PMID: 28349362 DOI: 10.1007/s12020-017-1280-y] [Citation(s) in RCA: 251] [Impact Index Per Article: 31.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/21/2016] [Accepted: 02/25/2017] [Indexed: 12/13/2022]
Abstract
PURPOSE Androgenetic alopecia, commonly known as male pattern baldness, is the most common type of progressive hair loss disorder in men. The aim of this paper is to review recent advances in understanding the pathophysiology and molecular mechanism of androgenetic alopecia. METHODS Using the PubMed database, we conducted a systematic review of the literature, selecting studies published from 1916 to 2016. RESULTS The occurrence and development of androgenetic alopecia depends on the interaction of endocrine factors and genetic predisposition. Androgenetic alopecia is characterized by progressive hair follicular miniaturization, caused by the actions of androgens on the epithelial cells of genetically susceptible hair follicles in androgen-dependent areas. Although the exact pathogenesis of androgenetic alopecia remains to be clarified, research has shown that it is a polygenetic condition. Numerous studies have unequivocally identified two major genetic risk loci for androgenetic alopecia, on the X-chromosome AR⁄EDA2R locus and the chromosome 20p11 locus. CONCLUSIONS Candidate gene and genome-wide association studies have reported that single-nucleotide polymorphisms at different genomic loci are associated with androgenetic alopecia development. A number of genes determine the predisposition for androgenetic alopecia in a polygenic fashion. However, further studies are needed before the specific genetic factors of this polygenic condition can be fully explained.
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Affiliation(s)
- Francesca Lolli
- Department of Experimental Medicine, University of Rome "La Sapienza", Rome, Italy
| | - Francesco Pallotti
- Department of Experimental Medicine, University of Rome "La Sapienza", Rome, Italy
| | - Alfredo Rossi
- Department of Internal Medicine and Medical Specialties, University of Rome "La Sapienza", Rome, Italy
| | - Maria C Fortuna
- Department of Internal Medicine and Medical Specialties, University of Rome "La Sapienza", Rome, Italy
| | - Gemma Caro
- Department of Internal Medicine and Medical Specialties, University of Rome "La Sapienza", Rome, Italy
| | - Andrea Lenzi
- Department of Experimental Medicine, University of Rome "La Sapienza", Rome, Italy
| | - Andrea Sansone
- Department of Experimental Medicine, University of Rome "La Sapienza", Rome, Italy
| | - Francesco Lombardo
- Department of Experimental Medicine, University of Rome "La Sapienza", Rome, Italy.
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Chinnery HR, McMenamin PG, Dando SJ. Macrophage physiology in the eye. Pflugers Arch 2017; 469:501-515. [DOI: 10.1007/s00424-017-1947-5] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2016] [Revised: 01/29/2017] [Accepted: 01/31/2017] [Indexed: 10/20/2022]
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Shukla S, Tavakkoli F, Singh V, Sangwan VS. Mesenchymal stem cell therapy for corneal diseases. Expert Opin Orphan Drugs 2016. [DOI: 10.1080/21678707.2016.1215906] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Affiliation(s)
- Sachin Shukla
- Prof. Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, Tej Kohli Cornea Institute, L.V. Prasad Eye Institute, Hyderabad, India
| | - Fatemeh Tavakkoli
- Prof. Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, Tej Kohli Cornea Institute, L.V. Prasad Eye Institute, Hyderabad, India
- Centre for Genetic Disorders, Banaras Hindu University, Varanasi, India
| | - Vivek Singh
- Prof. Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, Tej Kohli Cornea Institute, L.V. Prasad Eye Institute, Hyderabad, India
| | - Virender Singh Sangwan
- Prof. Brien Holden Eye Research Centre, Centre for Ocular Regeneration, Hyderabad Eye Research Foundation, Tej Kohli Cornea Institute, L.V. Prasad Eye Institute, Hyderabad, India
- Srujana-Center for Innovation, Tej Kohli Cornea Institute, L. V. Prasad Eye Institute, Hyderabad, India
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Yu T, Rajendran V, Griffith M, Forrester JV, Kuffová L. High-risk corneal allografts: A therapeutic challenge. World J Transplant 2016; 6:10-27. [PMID: 27011902 PMCID: PMC4801785 DOI: 10.5500/wjt.v6.i1.10] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/03/2015] [Revised: 10/03/2015] [Accepted: 12/04/2015] [Indexed: 02/05/2023] Open
Abstract
Corneal transplantation is the most common surgical procedure amongst solid organ transplants with a high survival rate of 86% at 1-year post-grafting. This high success rate has been attributed to the immune privilege of the eye. However, mechanisms originally thought to promote immune privilege, such as the lack of antigen presenting cells and vessels in the cornea, are challenged by recent studies. Nevertheless, the immunological and physiological features of the cornea promoting a relatively weak alloimmune response is likely responsible for the high survival rate in “low-risk” settings. Furthermore, although corneal graft survival in “low-risk” recipients is favourable, the prognosis in “high-risk” recipients for corneal graft is poor. In “high-risk” grafts, the process of indirect allorecognition is accelerated by the enhanced innate and adaptive immune responses due to pre-existing inflammation and neovascularization of the host bed. This leads to the irreversible rejection of the allograft and ultimately graft failure. Many therapeutic measures are being tested in pre-clinical and clinical studies to counter the immunological challenge of “high-risk” recipients. Despite the prevailing dogma, recent data suggest that tissue matching together with use of systemic immunosuppression may increase the likelihood of graft acceptance in “high-risk” recipients. However, immunosuppressive drugs are accompanied with intolerance/side effects and toxicity, and therefore, novel cell-based therapies are in development which target host immune cells and restore immune homeostasis without significant side effect of treatment. In addition, developments in regenerative medicine may be able to solve both important short comings of allotransplantation: (1) graft rejection and ultimate graft failure; and (2) the lack of suitable donor corneas. The advances in technology and research indicate that wider therapeutic choices for patients may be available to address the worldwide problem of corneal blindness in both “low-risk” and “high-risk” hosts.
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Funderburgh JL, Funderburgh ML, Du Y. Stem Cells in the Limbal Stroma. Ocul Surf 2016; 14:113-20. [PMID: 26804252 DOI: 10.1016/j.jtos.2015.12.006] [Citation(s) in RCA: 83] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2015] [Revised: 12/16/2015] [Accepted: 12/24/2015] [Indexed: 12/13/2022]
Abstract
The corneal stroma contains a population of mesenchymal cells subjacent to the limbal basement membrane with characteristics of adult stem cells. These 'niche cells' support limbal epithelial stem cell viability. In culture by themselves, the niche cells display a phenotype typical of mesenchymal stem cells. These stromal stem cells exhibit a potential to differentiate to multiple cell types, including keratocytes, thus providing an abundant source of these rare cells for experimental and bioengineering applications. Stromal stem cells have also shown the ability to remodel pathological stromal tissue, suppressing inflammation and restoring transparency. Because stromal stem cells can be obtained by biopsy, they offer a potential for autologous stem cell treatment for stromal opacities. This review provides an overview of the status of work on this interesting cell population.
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Affiliation(s)
- James L Funderburgh
- Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213.
| | - Martha L Funderburgh
- Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
| | - Yiqin Du
- Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
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Shankar SP, Griffith M, Forrester JV, Kuffová L. Dendritic cells and the extracellular matrix: A challenge for maintaining tolerance/homeostasis. World J Immunol 2015; 5:113-130. [DOI: 10.5411/wji.v5.i3.113] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Revised: 09/18/2015] [Accepted: 11/11/2015] [Indexed: 02/05/2023] Open
Abstract
The importance of the extracellular matrix (ECM) in contributing to structural, mechanical, functional and tissue-specific features in the body is well appreciated. While the ECM was previously considered to be a passive bystander, it is now evident that it plays active, dynamic and flexible roles in shaping cell survival, differentiation, migration and death to varying extents depending on the specific site in the body. Dendritic cells (DCs) are recognized as potent antigen presenting cells present in many tissues and in blood, continuously scrutinizing the microenvironment for antigens and mounting local and systemic host responses against harmful agents. DCs also play pivotal roles in maintaining homeostasis to harmless self-antigens, critical for preventing autoimmunity. What is less understood are the complex interactions between DCs and the ECM in maintaining this balance between steady-state tissue residence and DC activation during inflammation. DCs are finely tuned to inflammation-induced variations in fragment length, accessible epitopes and post-translational modifications of individual ECM components and correspondingly interpret these changes appropriately by adjusting their profiles of cognate binding receptors and downstream immune activation. The successful design and composition of novel ECM-based mimetics in regenerative medicine and other applications rely on our improved understanding of DC-ECM interplay in homeostasis and the challenges involved in maintaining it.
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Sidney LE, Branch MJ, Dua HS, Hopkinson A. Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells. Cytotherapy 2015; 17:1706-22. [PMID: 26454751 DOI: 10.1016/j.jcyt.2015.08.003] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2015] [Revised: 08/06/2015] [Accepted: 08/19/2015] [Indexed: 12/12/2022]
Abstract
BACKGROUND AIMS The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. METHODS Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. RESULTS All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. CONCLUSIONS Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy.
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Affiliation(s)
- Laura E Sidney
- Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham, Nottingham, United Kingdom.
| | - Matthew J Branch
- Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham, Nottingham, United Kingdom
| | - Harminder S Dua
- Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham, Nottingham, United Kingdom
| | - Andrew Hopkinson
- Academic Ophthalmology, Division of Clinical Neuroscience, Queen's Medical Centre Campus, University of Nottingham, Nottingham, United Kingdom
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Sidney LE, Branch MJ, Dunphy SE, Dua HS, Hopkinson A. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells 2015; 32:1380-9. [PMID: 24497003 PMCID: PMC4260088 DOI: 10.1002/stem.1661] [Citation(s) in RCA: 613] [Impact Index Per Article: 61.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2013] [Revised: 12/20/2013] [Accepted: 01/15/2014] [Indexed: 12/11/2022]
Abstract
CD34 is a transmembrane phosphoglycoprotein, first identified on hematopoietic stem and progenitor cells. Clinically, it is associated with the selection and enrichment of hematopoietic stem cells for bone marrow transplants. Due to these historical and clinical associations, CD34 expression is almost ubiquitously related to hematopoietic cells, and it is a common misconception that CD34-positive (CD34+) cells in nonhematopoietic samples represent hematopoietic contamination. The prevailing school of thought states that multipotent mesenchymal stromal cells (MSC) do not express CD34. However, strong evidence demonstrates CD34 is expressed not only by MSC but by a multitude of other nonhematopoietic cell types including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors. In many cases, the CD34+ cells represent a small proportion of the total cell population and also indicate a distinct subset of cells with enhanced progenitor activity. Herein, we explore common traits between cells that express CD34, including associated markers, morphology and differentiation potential. We endeavor to highlight key similarities between CD34+ cells, with a focus on progenitor activity. A common function of CD34 has yet to be elucidated, but by analyzing and understanding links between CD34+ cells, we hope to be able to offer an insight into the overlapping properties of cells that express CD34. Stem Cells2014;32:1380–1389
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Affiliation(s)
- Laura E Sidney
- Academic Ophthalmology, Division of Clinical Neuroscience, University of Nottingham, Queen's Medical Centre Campus, Nottingham, United Kingdom
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Seo Y, Kim MK, Lee JH, Chang EJ, Kim EK, Lee HK. Expression of Lymphangiogenic Markers in Rejected Human Corneal Buttons after Penetrating Keratoplasty. Curr Eye Res 2014; 40:902-12. [DOI: 10.3109/02713683.2014.969809] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Serratrice N, Cubizolle A, Ibanes S, Mestre-Francés N, Bayo-Puxan N, Creyssels S, Gennetier A, Bernex F, Verdier JM, Haskins ME, Couderc G, Malecaze F, Kalatzis V, Kremer EJ. Corrective GUSB transfer to the canine mucopolysaccharidosis VII cornea using a helper-dependent canine adenovirus vector. J Control Release 2014; 181:22-31. [PMID: 24607662 DOI: 10.1016/j.jconrel.2014.02.022] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2013] [Revised: 02/21/2014] [Accepted: 02/24/2014] [Indexed: 12/31/2022]
Abstract
Corneal transparency is maintained, in part, by specialized fibroblasts called keratocytes, which reside in the fibrous lamellae of the stroma. Corneal clouding, a condition that impairs visual acuity, is associated with numerous diseases, including mucopolysaccharidosis (MPS) type VII. MPS VII is due to deficiency in β-glucuronidase (β-glu) enzymatic activity, which leads to accumulation of glycosaminoglycans (GAGs), and secondary accumulation of gangliosides. Here, we tested the efficacy of canine adenovirus type 2 (CAV-2) vectors to transduce keratocyte in vivo in mice and nonhuman primates, and ex vivo in dog and human corneal explants. Following efficacy studies, we asked if we could treat corneal clouding by the injection a helper-dependent (HD) CAV-2 vector (HD-RIGIE) harboring the human cDNA coding for β-glu (GUSB) in the canine MPS VII cornea. β-Glu activity, GAG content, and lysosome morphology and physiopathology were analyzed. We found that HD-RIGIE injections efficiently transduced coxsackievirus adenovirus receptor-expressing keratocytes in the four species and, compared to mock-injected controls, improved the pathology in the canine MPS VII cornea. The key criterion to corrective therapy was the steady controlled release of β-glu and its diffusion throughout the collagen-dense stroma. These data support the continued evaluation of HD CAV-2 vectors to treat diseases affecting corneal keratocytes.
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Affiliation(s)
- Nicolas Serratrice
- Institut de Génétique Moléculaire de Montpellier, CNRS 5535, Montpellier, France; Université de Montpellier I, Montpellier, France; Université Montpellier 2, Montpellier, France
| | - Aurelie Cubizolle
- Institut de Génétique Moléculaire de Montpellier, CNRS 5535, Montpellier, France; Université de Montpellier I, Montpellier, France; Université Montpellier 2, Montpellier, France
| | - Sandy Ibanes
- Institut de Génétique Moléculaire de Montpellier, CNRS 5535, Montpellier, France; Université de Montpellier I, Montpellier, France; Université Montpellier 2, Montpellier, France
| | - Nadine Mestre-Francés
- Université Montpellier 2, Montpellier, France; Inserm U710, Montpellier, France; Ecole Pratique des Hautes Etudes, Paris, France
| | - Neus Bayo-Puxan
- Institut de Génétique Moléculaire de Montpellier, CNRS 5535, Montpellier, France; Université de Montpellier I, Montpellier, France; Université Montpellier 2, Montpellier, France
| | - Sophie Creyssels
- Institut de Génétique Moléculaire de Montpellier, CNRS 5535, Montpellier, France; Université de Montpellier I, Montpellier, France; Université Montpellier 2, Montpellier, France
| | - Aurelie Gennetier
- Institut de Génétique Moléculaire de Montpellier, CNRS 5535, Montpellier, France; Université de Montpellier I, Montpellier, France; Université Montpellier 2, Montpellier, France
| | - Florence Bernex
- Institut Régional du Cancer Montpellier, Inserm U896, Montpellier, France
| | - Jean-Michel Verdier
- Université Montpellier 2, Montpellier, France; Inserm U710, Montpellier, France; Ecole Pratique des Hautes Etudes, Paris, France
| | - Mark E Haskins
- Department of Pathobiology, School of Veterinary Medicine, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Guilhem Couderc
- Tissue Bank, Centre Hospitalier Régional Universitaire de Montpellier, Montpellier, France
| | - Francois Malecaze
- Inserm U563, Toulouse, France; Departement d'Ophtalmologie, Hôpital Purpan, Toulouse, France
| | - Vasiliki Kalatzis
- Institut de Génétique Moléculaire de Montpellier, CNRS 5535, Montpellier, France; Université de Montpellier I, Montpellier, France; Université Montpellier 2, Montpellier, France
| | - Eric J Kremer
- Institut de Génétique Moléculaire de Montpellier, CNRS 5535, Montpellier, France; Université de Montpellier I, Montpellier, France; Université Montpellier 2, Montpellier, France.
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Schewitz-Bowers LP, Lee RWJ, Dick AD. Immune mechanisms of intraocular inflammation. EXPERT REVIEW OF OPHTHALMOLOGY 2014. [DOI: 10.1586/eop.09.68] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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Hashmani K, Branch MJ, Sidney LE, Dhillon PS, Verma M, McIntosh OD, Hopkinson A, Dua HS. Characterization of corneal stromal stem cells with the potential for epithelial transdifferentiation. Stem Cell Res Ther 2013; 4:75. [PMID: 23800436 PMCID: PMC4058700 DOI: 10.1186/scrt226] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2013] [Accepted: 06/04/2013] [Indexed: 12/12/2022] Open
Abstract
INTRODUCTION The corneal stroma is being increasingly recognized as a repository for stem cells. Like the limbal and endothelial niches, stromal stem cells often reside in the peripheral cornea and limbus. These peripheral and limbal corneal stromal cells (PLCSCs) are known to produce mesenchymal stem cells in vitro. Recently, a common corneal stromal and epithelial progenitor was hinted at. This study aims to examine the stem cell potential of corneal stromal cells and to investigate their epithelial transdifferentiation ability. METHODS PLCSCs were grown in traditional Dulbecco modified Eagle medium (DMEM)-based keratocyte culture medium and an M199-based medium and analyzed for a profile of cell-surface markers by using flow cytometry and differentiated into mesenchymal phenotypes analyzed with quantitative polymerase chain reaction (qPCR) and histologic staining. PLCSCs in M199 were subsequently divided into subpopulations based on CD34 and CD105 expression by using fluorescence- activated cell sorting (FACS). Subpopulations were characterized by marker profile and mesenchymal differentiation ability. Both whole PLCSCs and subpopulations were also cultured for epithelial transdifferentiation. RESULTS Cells cultured in M199 demonstrated a more stem-like cell-surface marker profile, and the keratocyte marker CD34 was retained for several passages but absent in cells cultured in DMEM. Cells cultured in M199 also exhibited a greater mesenchymal differentiation potential, compared with DMEM. PLCSCs could be divided into CD34(+)CD105(+), CD34-CD105(+), and CD34-CD105- subpopulations, of which CD34(+)CD105(+) cells were the most stemlike with regard to marker expression and mesenchymal differentiation potential. Subpopulations of PLCSCs exhibited differing abilities to transdifferentiate into epithelial phenotypes. Cells that were initially CD34(+)CD105(+) showed the greatest differentiation potential, producing CK3(+) and CK19(+) cells, and expressed a range of both epithelial progenitor (HES1, FRZB1, DCT, SOD2, ABCG2, CDH1, KRT19) and terminally differentiated (DSG3, KRT3, KRT12, KRT24) genes. CONCLUSIONS Culture medium has a significant effect on the phenotype and differentiation capacity of PLCSCs. The stroma contains a heterogeneous cell population in which we have identified CD34(+) cells as a stem cell population with a capacity for mesenchymal and epithelial differentiation.
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Galectin-9-mediated protection from allo-specific T cells as a mechanism of immune privilege of corneal allografts. PLoS One 2013; 8:e63620. [PMID: 23667648 PMCID: PMC3646846 DOI: 10.1371/journal.pone.0063620] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2013] [Accepted: 04/04/2013] [Indexed: 12/12/2022] Open
Abstract
The eye is an immune-privileged organ, and corneal transplantation is therefore one of the most successful organ transplantation. The immunosuppressive intraocular microenvironment is known as one of the mechanisms underlying immune privilege in the eye. T-cell immunoglobulin and mucin domain (Tim)-3 is a regulatory molecule for T-cell function, and galectin (Gal)-9 is a Tim-3 ligand. We investigated the role of this pathway in establishing the immune-privileged status of corneal allografts in mice. Gal-9 is constitutively expressed on the corneal epithelium, endothelium and iris-ciliary body in normal mouse eyes and eyes bearing surviving allografts, and Tim-3 was expressed on CD8 T cells infiltrating the allografts. Allograft survival in recipients treated with anti-Tim-3 monoclonal antibody (mAb) or anti-Gal-9 mAb was significantly shorter than that in control recipients. In vitro, destruction of corneal endothelial cells by allo-reactive T cells was enhanced when the cornea was pretreated with anti-Gal-9 mAb. Blockade of Tim-3 or Gal-9 did not abolish anterior chamber-associated immune deviation. We propose that constitutive expression of Gal-9 plays an immunosuppressive role in corneal allografts. Gal-9 expressed on corneal endothelial cells protects them from destruction by allo-reactive T cells within the cornea.
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Forrester JV, Steptoe RJ, Klaska IP, Martin-Granados C, Dua HS, Degli-Esposti MA, Wikstrom ME. Cell-based therapies for ocular inflammation. Prog Retin Eye Res 2013; 35:82-101. [PMID: 23542232 DOI: 10.1016/j.preteyeres.2013.02.002] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2012] [Revised: 01/31/2013] [Accepted: 02/01/2013] [Indexed: 12/13/2022]
Abstract
Since the plasticity and the potential for re-programming cells has become widely accepted, there has been great interest in cell-based therapies. These are being applied to a range of diseases, not least ocular diseases, where it is assumed that there is a reduced risk of immune rejection although this may be more perceived than real. There are two broad classes of cell-based therapies: those aimed at restoring structure and function of specific tissues and cells; and those directed towards restoring immunological homeostasis by controlling the damaging effects of inflammatory disease. Stem cells of all types represent the first group and prototypically have been used with the aim of regenerating failing cells. In contrast, immune cells have been suggested as potential modulators of inflammation. However, there is functional overlap in these two applications, with some types of stem cells, such as mesenchymal stem cells, demonstrating a potent immunomodulatory effect. This review summarises recent information on cell based therapies for ocular disease, with special emphasis on ocular inflammatory disease, and explores current uses, potential and limitations.
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Affiliation(s)
- John V Forrester
- Immunology Program, Centre for Ophthalmology and Visual Science, The University of Western Australia, Perth, Western Australia, Australia.
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Vitova A, Kuffová L, Klaska IP, Holan V, Cornall RJ, Forrester JV. The high-risk corneal regraft model: a justification for tissue matching in humans. Transpl Int 2013; 26:453-61. [PMID: 23398177 DOI: 10.1111/tri.12055] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2012] [Revised: 06/18/2012] [Accepted: 12/14/2012] [Indexed: 12/17/2022]
Abstract
Models of high-risk corneal graft rejection involve neovascularization induced via innate immune responses, e.g., suture-mediated trauma. We describe a model of high-risk corneal graft rejection using corneal graft donor-recipient pairing based on a single-antigen disparity. Donor corneas from transgenic mice on B10.BR (H-2k ) background, in which hen-egg lysozyme (HEL) as a membrane-bound antigen (mHEL) was expressed under the major histocompatibility complex (MHC) class I promoter (KLK-mHEL, H-2k), were transplanted into wild type B10.BR recipient mice. Unmanipulated wild type recipient mice rejected KLK-mHEL grafts (39%) slowly over 50-60 days. Graft rejection incidence was maximized (100%) and tempo accelerated (27 days) by priming with HEL-pulsed syngeneic dendritic cells and less so by increasing T-cell precursor frequency. Rejection also reached maximum levels (100%) and tempo (3-8 days) when mice which had rejected a first graft ('rejectors') were regrafted, and was associated with induction of HEL-specific memory T cells. In contrast, 'acceptors' rejected a second graft at rates and tempo similar to naïve mice. These data reveal the importance of (i) donor MHC antigens as alloantigens for indirect recognition, (ii) alloantigen-specific memory in high-risk graft rejection involving regrafts, and (iii) suggest a role for tissue matching in human corneal graft to avoid sensitization to donor MHC antigens.
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Affiliation(s)
- Andrea Vitova
- Section of Immunology and Infection, Division of Applied Medicine, University of Aberdeen, Aberdeen, UK
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Saban DR, Calder V, Kuo CH, Reyes NJ, Dartt DA, Ono SJ, Niederkorn JY. New twists to an old story: novel concepts in the pathogenesis of allergic eye disease. Curr Eye Res 2013; 38:317-30. [PMID: 23281793 DOI: 10.3109/02713683.2012.747617] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
The prevalence of allergy is rising globally at a very significant rate, which is currently at 20-40% of individuals in westernized nations. In the eye, allergic conditions can take on the acute form such as in seasonal and perennial allergic conjunctivitis, or a more severe and debilitating chronic form such as in vernal and atopic keratoconjunctivitis. Indeed, some key aspects of allergic eye disease pathophysiology are understood, such as the role of mast cells in the acute allergic reaction, and the contribution of eosinophils in late-onset and chronic allergy. However, recent developments in animal models and clinical studies have uncovered new and important roles for previously underappreciated players, including chemokine receptors on ocular surface dendritic cells such as CCR7, the contribution of conjunctival epithelium to immunity, histamine and leukotriene receptors on conjunctival goblet cells and a role for mast cells in late-onset manifestations. Furthermore, recent work in animal models has delineated the contribution of IL-4 in the increased incidence of corneal graft rejection in hosts with allergic conjunctivitis. Recent studies such as these mean that conventional paradigms and concepts should be revisited. The aim of this review is to highlight some of the most recent advances and insights on newly appreciated players in the pathogenesis of allergic eye disease.
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Affiliation(s)
- Daniel R Saban
- Department of Ophthalmology, Duke University School of Medicine, Durham, NC 27710, USA.
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Kunishige T, Hori J. Immune privilege as new therapeutic strategies for success of corneal transplantation. Inflamm Regen 2013. [DOI: 10.2492/inflammregen.33.274] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
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Abstract
The cornea is a tough transparent tissue admitting and focusing light in the eye. More than 90% of the cornea is stroma, a highly organized, transparent connective tissue maintained by keratocytes, quiescent mesenchymal cells of neural crest origin. A small population of cells in the mammalian stroma displays properties of mesenchymal stem cells, including clonal growth, multipotent differentiation, and expression of an array of stem cell-specific markers. Unlike keratocytes, the corneal stromal stem cells (CSSCs) undergo extensive expansion in vitro without loss of the ability to adopt a keratocyte phenotype. Several lines of evidence suggest CSSCs to be of neural crest lineage and not from bone marrow. CSSCs are localized in the anterior peripheral (limbal) stroma near to stem cells of the corneal epithelium. CSSCs may function to support potency of the epithelial stem cells in their unique limbal niche. On the other hand, little information is available documenting a role for CSSCs in vivo in stromal wound healing or regeneration. In vitro CSSCs reproduce the highly organized connective tissue of the stroma, demonstrating a potential use of these cells in tissue bioengineering. Direct introduction of CSSCs into the corneal stroma generated transparent tissue in a mouse model of corneal opacity. Human CSSCs injected into mice corneas did not elicit immune rejection over an extended period of time. The CSSCs therefore appear offer an opportunity to develop cell- and tissue-based therapies for irreversible corneal blindness, conditions affecting more than 10 million individuals worldwide.
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Affiliation(s)
- Niveditha Pinnamaneni
- Department of Ophthalmology, UPMC Eye Center, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
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Forrester JV, Xu H. Good news-bad news: the Yin and Yang of immune privilege in the eye. Front Immunol 2012; 3:338. [PMID: 23230433 PMCID: PMC3515883 DOI: 10.3389/fimmu.2012.00338] [Citation(s) in RCA: 106] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2012] [Accepted: 10/23/2012] [Indexed: 12/27/2022] Open
Abstract
The eye and the brain are prototypical tissues manifesting immune privilege (IP) in which immune responses to foreign antigens, particularly alloantigens are suppressed, and even completely inhibited. Explanations for this phenomenon are numerous and mostly reflect our evolving understanding of the molecular and cellular processes underpinning immunological responses generally. IP is now viewed as a property of many tissues and the level of expression of IP varies not only with the tissue but with the nature of the foreign antigen and changes in the limited conditions under which privilege can operate as a mechanism of immunological tolerance. As a result, IP functions normally as a homeostatic mechanism preserving normal function in tissues, particularly those with highly specialized function and limited capacity for renewal such as the eye and brain. However, IP is relatively easily bypassed in the face of a sufficiently strong immunological response, and the privileged tissues may be at greater risk of collateral damage because its natural defenses are more easily breached than in a fully immunocompetent tissue which rapidly rejects foreign antigen and restores integrity. This two-edged sword cuts its swathe through the eye: under most circumstances, IP mechanisms such as blood-ocular barriers, intraocular immune modulators, induction of T regulatory cells, lack of lymphatics, and other properties maintain tissue integrity; however, when these are breached, various degrees of tissue damage occur from severe tissue destruction in retinal viral infections and other forms of uveoretinal inflammation, to less severe inflammatory responses in conditions such as macular degeneration. Conversely, ocular IP and tumor-related IP can combine to permit extensive tumor growth and increased risk of metastasis thus threatening the survival of the host.
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Affiliation(s)
- John V. Forrester
- Laboratory of Immunology, Lion’s Eye Institute, University of Western AustraliaPerth, WA, Australia
- Ocular Immunology Laboratory, Section of Immunology and Infection, Institute of Medical Sciences, University of AberdeenAberdeen, UK
| | - Heping Xu
- Laboratory of Immunology, Lion’s Eye Institute, University of Western AustraliaPerth, WA, Australia
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Liu H, Zhang J, Liu CY, Hayashi Y, Kao WWY. Bone marrow mesenchymal stem cells can differentiate and assume corneal keratocyte phenotype. J Cell Mol Med 2012; 16:1114-24. [PMID: 21883890 PMCID: PMC4365890 DOI: 10.1111/j.1582-4934.2011.01418.x] [Citation(s) in RCA: 74] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
It remains elusive as to what bone marrow (BM) cell types infiltrate into injured and/or diseased tissues and subsequently differentiate to assume the phenotype of residential cells, for example, neurons, cardiac myocytes, keratocytes, etc., to repair damaged tissue. Here, we examined the possibility of whether BM cell invasion via circulation into uninjured and injured corneas could assume a keratocyte phenotype, using chimeric mice generated by transplantation of enhanced green fluorescent protein (EGFP)+ BM cells into keratocan null (Kera−/−) and lumican null (Lum−/−) mice. EGFP+ BM cells assumed dendritic cell morphology, but failed to synthesize corneal-specific keratan sulfate proteoglycans, that is KS-lumican and KS-keratocan. In contrast, some EGFP+ BM cells introduced by intrastromal transplantation assumed keratocyte phenotypes. Furthermore, BM cells were isolated from Kera-Cre/ZEG mice, a double transgenic mouse line in which cells expressing keratocan become EGFP+ due to the synthesis of Cre driven by keratocan promoter. Three days after corneal and conjunctival transplantations of such BM cells into Kera−/− mice, green keratocan positive cells were found in the cornea, but not in conjunctiva. It is worthy to note that transplanted BM cells were rejected in 4 weeks. MSC isolated from BM were used to examine if BM mesenchymal stem cells (BM-MSC) could assume keratocyte phenotype. When BM-MSC were intrastromal-transplanted into Kera−/− mice, they survived in the cornea without any immune and inflammatory responses and expressed keratocan in Kera−/− mice. These observations suggest that corneal intrastromal transplantation of BM-MSC may be an effective treatment regimen for corneal diseases involving dysfunction of keratocytes.
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Affiliation(s)
- Hongshan Liu
- Department of Ophthalmology, Edith Crawley Vision Research Center, University of Cincinnati, Cincinnati, OH, USA.
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Gonzalez JM, Heur M, Tan JCH. Two-photon immunofluorescence characterization of the trabecular meshwork in situ. Invest Ophthalmol Vis Sci 2012; 53:3395-404. [PMID: 22531697 DOI: 10.1167/iovs.11-8570] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
PURPOSE To develop an in situ model to study biological responses and glaucoma pathology in the human trabecular meshwork (TM). Characteristic TM cell- and glaucoma-associated markers were localized in situ in relation to the tissue's autofluorescent structural extracellular matrix (ECM) by two-photon excitation fluorescence optical sectioning (TPEF). METHODS Human donor corneoscleral (CS) tissue containing the intact aqueous drainage tract was incubated with dexamethasone (Dex) or TGF-β1, and immunostained for epifluorescence (EF) microscopy with antibodies to myocilin and alpha smooth muscle (α-SMA). Separate specimens were labeled for Type-IV collagen and fibronectin. Nuclei were stained with Hoechst 33342. Multimodal TPEF was used to visualize EF, intravital dyes, and autofluorescence (AF) in situ. Three-dimensional (3D) localization of fluorescence within the TM was analyzed using reconstruction software. RESULTS Autofluorescent beams, perforated sheets, and fibers, consistent with the uveal (UV), CS, and juxtacanalicular (JCT) meshwork, respectively, were captured at different depths of the TM. Type-IV collagen EF distinctly outlined the AF beams in a location consistent with basement membrane. Fibronectin EF showed a diffuse reticular pattern throughout the TM. TGF-β1-induced α-SMA expression, which was distributed perinuclearly in cells among autofluorescent structures. Dex-induced myocilin expression had both cytosolic and extracellular distributions. CONCLUSIONS The authors have localized markers that are characteristic of TM cells and relevant to glaucoma pathogenesis in situ using multimodal TPEF without conventional histological embedding and sectioning. Protein expression was inducible in situ and could be analyzed with respect to cells and the ECM within the 3D environment of the human TM.
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Affiliation(s)
- Jose M Gonzalez
- Doheny Eye Institute and Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA
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Hippert C, Ibanes S, Serratrice N, Court F, Malecaze F, Kremer EJ, Kalatzis V. Corneal transduction by intra-stromal injection of AAV vectors in vivo in the mouse and ex vivo in human explants. PLoS One 2012; 7:e35318. [PMID: 22523585 PMCID: PMC3327666 DOI: 10.1371/journal.pone.0035318] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2011] [Accepted: 03/14/2012] [Indexed: 12/13/2022] Open
Abstract
The cornea is a transparent, avascular tissue that acts as the major refractive surface of the eye. Corneal transparency, assured by the inner stroma, is vital for this role. Disruption in stromal transparency can occur in some inherited or acquired diseases. As a consequence, light entering the eye is blocked or distorted, leading to decreased visual acuity. Possible treatment for restoring transparency could be via viral-based gene therapy. The stroma is particularly amenable to this strategy due to its immunoprivileged nature and low turnover rate. We assayed the potential of AAV vectors to transduce keratocytes following intra-stromal injection in vivo in the mouse cornea and ex vivo in human explants. In murine and human corneas, we transduced the entire stroma using a single injection, preferentially targeted keratocytes and achieved long-term gene transfer (up to 17 months in vivo in mice). Of the serotypes tested, AAV2/8 was the most promising for gene transfer in both mouse and man. Furthermore, transgene expression could be transiently increased following aggression to the cornea.
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Affiliation(s)
- Claire Hippert
- Institut de Génétique Moléculaire de Montpellier, CNRS, Montpellier, France
- Universités Montpellier I & II, Montpellier, France
| | - Sandy Ibanes
- Institut de Génétique Moléculaire de Montpellier, CNRS, Montpellier, France
- Universités Montpellier I & II, Montpellier, France
| | - Nicolas Serratrice
- Institut de Génétique Moléculaire de Montpellier, CNRS, Montpellier, France
- Universités Montpellier I & II, Montpellier, France
| | - Franck Court
- Institut de Génétique Moléculaire de Montpellier, CNRS, Montpellier, France
- Universités Montpellier I & II, Montpellier, France
| | - François Malecaze
- Inserm U563, Toulouse, France
- Département d'Ophtalmologie, Hôpital Purpan, Toulouse, France
| | - Eric J. Kremer
- Institut de Génétique Moléculaire de Montpellier, CNRS, Montpellier, France
- Universités Montpellier I & II, Montpellier, France
| | - Vasiliki Kalatzis
- Institut de Génétique Moléculaire de Montpellier, CNRS, Montpellier, France
- Universités Montpellier I & II, Montpellier, France
- * E-mail:
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Chinnery HR, McLenachan S, Binz N, Sun Y, Forrester JV, Degli-Esposti MA, Pearlman E, McMenamin PG. TLR9 ligand CpG-ODN applied to the injured mouse cornea elicits retinal inflammation. THE AMERICAN JOURNAL OF PATHOLOGY 2011; 180:209-20. [PMID: 22085974 DOI: 10.1016/j.ajpath.2011.09.041] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/28/2011] [Revised: 09/11/2011] [Accepted: 09/14/2011] [Indexed: 11/15/2022]
Abstract
During bacterial and viral infections, unmethylated CpG-DNA released by proliferating and dying microbes is recognized by toll-like receptor (TLR) 9 in host cells, initiating innate immune responses. Many corneal infections occur secondary to epithelial breaches and represent a major cause of vision impairment and blindness globally. To mimic this clinical situation, we investigated mechanisms of TLR9 ligand-induced corneal inflammation in mice after epithelial debridement. Application of CpG oligodeoxynucleotides (ODNs) resulted in neutrophil and macrophage infiltration to the cornea and loss of transparency. By 6 hours after CpG-ODN administration, TLR9 mRNA was increased in the cornea and retina. In vivo clinical examination at 24 hours revealed inflammatory infiltrates in the vitreous and retina, which were confirmed ex vivo to be neutrophils and macrophages, along with activated resident microglia. CpG-ODN-induced intraocular inflammation was abrogated in TLR9(-/-) and macrophage-depleted mice. Bone marrow reconstitution of irradiated TLR9(-/-) mice with TLR9(+/+) bone marrow led to restored corneal inflammatory responses to CpG-ODN. Fluorescein isothiocyanate-CpG-ODN rapidly penetrated the cornea and ocular media to reach the retina, where it was present within CD68(+) retinal macrophages and microglia. These data show that topically applied CpG-ODN induces intraocular inflammation owing to TLR9 activation of monocyte-lineage cells. These novel findings indicate that microbial CpG-DNA released during bacterial and/or viral keratitis can cause widespread inflammation within the eye, including the retina.
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Affiliation(s)
- Holly R Chinnery
- Department of Anatomy and Developmental Biology, School of Biomedical Sciences, Monash University, Clayton, VIC, Australia
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Hattori T, Chauhan SK, Lee H, Ueno H, Dana R, Kaplan DH, Saban DR. Characterization of Langerin-expressing dendritic cell subsets in the normal cornea. Invest Ophthalmol Vis Sci 2011; 52:4598-604. [PMID: 21482644 DOI: 10.1167/iovs.10-6741] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
PURPOSE In addition to Langerhans cells (LCs), other dendritic cells (CD11c(+)) have recently been shown to express Langerin (c-type lectin). In skin, (non-LC) Langerin+ dendritic cells initiate adaptive immunity. However, whether such dendritic cells (DC) reside in the cornea, an immune-privileged tissue, is unknown. METHODS Normal C57BL/6 corneas were harvested for qRT-PCR analyses of Langerin expression in the epithelium versus stroma. Immunohistochemistry for Langerin was also performed. Single-cell preparations of epithelium versus stroma were FACS analyzed for CD11c, CD11b, and CD103 expression. Fluorescence microscopy of corneas from muLangerin-eGFP mice (in which all CD11c(+) Langerin+ cells express eGFP), huLangerin-DTA mice (only LCs are constitutively deleted), and huLangerin-Cre eYFP-flox (only LCs express eYFP) was performed. RESULTS qRT-PCR, immunohistochemistry, and FACS analysis identified CD11c(+) Langerin+ cells in the epithelium and stroma. Similarly, corneas of muLangerin-eGFP mice contained eGFP+ cells in the epithelium and stroma. However, FACS analysis indicated phenotypically differing CD11c(+) Langerin+ populations in the epithelium (CD11b(low)CD103(low)) versus stroma (CD11b(+)CD103(low)). Additionally, corneas from huLangerin-DTA mice were devoid of Langerin+ cells in the epithelium but were detectable in the stroma. In corneas from huLangerin-Cre eYFP-flox, eYFP+ cells were detectable in the epithelium but not in the stroma. CONCLUSIONS The normal corneal epithelium is endowed with CD11c(+) Langerin+ cells that are LCs, whereas the stroma is endowed with a separate population of (non-LC) Langerin+ DCs. These findings should henceforth facilitate the examination of Langerin-expressing DC subsets in the immunopathogeneses of conditions such as keratoconjunctivitis sicca, allergic keratoconjunctivitis, and corneal allograft rejection.
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Affiliation(s)
- Takaaki Hattori
- Schepens Eye Research Institute, Boston, Massachusetts 02117, USA
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A perfluoropolyether corneal inlay for the correction of refractive error. Biomaterials 2011; 32:3158-65. [PMID: 21306775 DOI: 10.1016/j.biomaterials.2011.01.047] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2010] [Accepted: 01/12/2011] [Indexed: 11/24/2022]
Abstract
This study assessed the long-term biological response of a perfluoropolyether-based polymer developed as a corneal inlay to correct refractive error. The polymer formulation met chemical and physical specifications and was non-cytotoxic when tested using standard in vitro techniques. It was cast into small microporous membranes that were implanted as inlays into corneas of rabbits (n = 5) and unsighted humans (n = 5 + 1 surgical control) which were monitored for up to 23 and 48 months respectively. Overall, the inlays were well tolerated during study period with the corneas remaining clear and holding a normal tear film and with no increased vascularisation or redness recorded. Inlays in three human corneas continued past 48 months without sequelae. Inlays in two human corneas were removed early due to small, focal erosions developing 5 and 24 months post-implantation. Polymer inlays maintained their integrity and corneal position for the study duration although the optical clarity of the inlays reduced slowly with time. Inlays induced corneal curvature changes in human subjects that showed stability with time and the refractive effect was reversed when the inlay was removed. Outcomes showed the potential of a perfluoropolyether inlay as a biologically acceptable corneal implant with which to provide stable correction of refractive error.
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Hori J, Vega JL, Masli S. Review of ocular immune privilege in the year 2010: modifying the immune privilege of the eye. Ocul Immunol Inflamm 2011; 18:325-33. [PMID: 20849282 DOI: 10.3109/09273948.2010.512696] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The original evidence for the existence of immunologically privileged sites in the body was based on the prolonged survival of genetically disparate transplanted tissue in the anterior chamber of the eye. The failure of the immune system to elicit an immune response in this and other such sites constitutes the hallmark of the immune privilege status. The remarkably successful field of corneal transplantation in clinical practice is undoubtedly associated with corneal immune privilege. Several investigations have addressed the regulatory mechanisms governing this phenomenon, which involves a complex interplay between multiple molecular and cellular pathways. Furthermore, the use of various transgenic mouse models has facilitated the identification of critical pathways, which upon disruption can modify the immune privileged status of the eye. Understanding these pathways not only reveals the mechanisms underlying various ocular inflammatory disease conditions, but also has clinical implications for the transplantation field and for the treatment of autoimmunity.
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Affiliation(s)
- Junko Hori
- Department of Ophthalmology, Nippon Medical School, Tokyo, Japan
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de Benito Llopis L, Drake P, Cañadas P, Hernández-Verdejo JL, Teus MA. Keratocyte density after laser-assisted subepithelial keratectomy with mitomycin C. Am J Ophthalmol 2010; 150:642-649.e1. [PMID: 20691417 DOI: 10.1016/j.ajo.2010.05.029] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2010] [Revised: 05/20/2010] [Accepted: 05/20/2010] [Indexed: 11/16/2022]
Abstract
PURPOSE To study the effects of laser-assisted subepithelial keratectomy (LASEK) with mitomycin C (MMC) on the keratocyte population. DESIGN Prospective, nonrandomized, interventional, comparative case series. METHODS Fifty-six eyes treated at Vissum Santa Hortensia, Madrid, Spain, were included in the study. We compared 28 eyes treated with LASEK with intraoperative 0.02% MMC versus 28 non-treated eyes. Keratocyte density was measured 3 months after the surgery in the anterior, mid, and posterior stroma and was compared with the corresponding layers in the control eyes. The anterior layer in the LASEK group was compared with 2 layers in the control group: the most anterior stromal layer and the 80 μm-deep layer, because that was the mean ablation depth performed in eyes that underwent LASEK. RESULTS We found a statistically significantly lower keratocyte population in the most anterior stromal layer after LASEK with MMC compared with both the most anterior stromal layer and the 80 μm-deep layer in controls. On the contrary, the treated group showed a significantly higher keratocyte density in both the mid stroma and the deep stroma. The comparison between the average densities through the entire cornea showed a significantly higher keratocyte population in the LASEK with MMC group. CONCLUSIONS LASEK with MMC seems to cause a decrease in the anterior stromal cells 3 months after the surgery compared with nonoperated corneas. There seems to be a compensating proliferation of keratocytes in the deeper corneal layers, suggesting that the ability of keratocytes to repopulate the cornea is maintained after the surgical procedure.
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Lu JM, Zhou ZY, Zhang XR, Li XL, Wang HF, Song XJ. A preliminary study of mesenchymal stem cell-like cells derived from murine corneal stroma. Graefes Arch Clin Exp Ophthalmol 2010; 248:1279-85. [PMID: 20390423 DOI: 10.1007/s00417-010-1367-0] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2010] [Revised: 03/08/2010] [Accepted: 03/11/2010] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Mesenchymal stem cells can be isolated from various tissues besides bone marrow and can differentiate into cells of three germ layers. Recent studies indicate that some cells in corneal stroma express stem cell markers and can also differentiate into chondrocytes and neurocytes. This study was carried out to investigate whether mesenchymal stem cells reside in the murine corneal stroma. METHODS Corneas of BALB/c mice were treated with collagenase digestion after the epithelium and endothelium were removed. Then the single cells were harvested and further identified by reverse transcription polymerase chain reaction (RT-PCR). After the immunophenotype of passage 2 corneal stroma-derived cells was analyzed by flow cytometry, attempts were made to differentiate these cells into adipocytes and osteocytes using conditioned medium. Following induction, cells were evaluated by RT-PCR, oil red O and Alizarin Red staining. RESULTS Isolated single cells were of stromal origin, not of epithelial or endothelial. Passage 2 corneal stroma-derived cells exhibited the spindle-shaped morphology and expressed CD29, CD90, CD105, and CD71; but were negative for CD34 and CD45. In addition, these cells showed the potentiality of differentiating into adipocytes and osteocytes, which was confirmed by RT-PCR and staining. CONCLUSION This study demonstrates the presence of mesenchymal stem cell-like cells in the murine corneal stroma. Further analysis of these cells will aid elucidation of the mechanisms of some keratopathies, and these cells may be a source for bioengineering of corneal tissue and for cell-based therapeutics.
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Affiliation(s)
- Jian-Min Lu
- Department of Ophthalmology, Third Hospital of Hebei Medical University, Ziqiang Road No. 139, Shijiazhuang, China
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Sun Y, Karmakar M, Roy S, Ramadan RT, Williams SR, Howell S, Shive CL, Han Y, Stopford CM, Rietsch A, Pearlman E. TLR4 and TLR5 on corneal macrophages regulate Pseudomonas aeruginosa keratitis by signaling through MyD88-dependent and -independent pathways. THE JOURNAL OF IMMUNOLOGY 2010; 185:4272-83. [PMID: 20826748 DOI: 10.4049/jimmunol.1000874] [Citation(s) in RCA: 106] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Pseudomonas aeruginosa is a major cause of blindness and visual impairment in the United States and worldwide. Using a murine model of keratitis in which abraded corneas are infected with P. aeruginosa parent and ΔfliC (aflagellar) strains 19660 and PAO1, we found that F4/80(+) macrophages were the predominant cell type in the cornea expressing TLR2, TLR4, and TLR5. Depletion of macrophages and dendritic cells using transgenic Mafia mice, in which Fas ligand is selectively activated in these cells, resulted in diminished cytokine production and cellular infiltration to the corneal stroma and unimpaired bacterial growth. TLR4(-/-) mice showed a similar phenotype postinfection with ΔfliC strains, whereas TLR4/5(-/-) mice were susceptible to corneal infection with parent strains. Bone marrow-derived macrophages stimulated with ΔfliC bacteria induced Toll/IL-1R intracellular domain (TIR)-containing adaptor inducing IFN-β (TRIF)-dependent phosphorylation of IFN regulatory factor 3 in addition to TIR-containing adaptor protein/MyD88-dependent phosphorylation of IκB and nuclear translocation of the p65 subunit of NFκB. Furthermore, TRIF(-/-) mice showed a similar phenotype as TLR4(-/-) mice in regulating only ΔfliC bacteria, whereas MyD88(-/-) mice were unable to clear parent or ΔfliC bacteria. Finally, IL-1R1(-/-) and IL-1α/β(-/-) mice were highly susceptible to infection. Taken together, these findings indicate that P. aeruginosa activates TLR4/5 on resident corneal macrophages, which signal through TRIF and TIR-containing adaptor protein/MyD88 pathways, leading to NF-κB translocation to the nucleus, transcription of CXCL1 and other CXC chemokines, recruitment of neutrophils to the corneal stroma, and subsequent bacterial killing and tissue damage. IL-1α and IL-1β are also produced, which activate an IL-1R1/MyD88-positive feedback loop in macrophages and IL-1R on other resident cells in the cornea.
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Affiliation(s)
- Yan Sun
- Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH 44106, USA
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