|
1
|
Gupta Y, Shanmugam C, K P, Mandal S, Tandon R, Sharma N. Pediatric keratoconus. Surv Ophthalmol 2025; 70:296-330. [PMID: 39396644 DOI: 10.1016/j.survophthal.2024.10.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2024] [Revised: 10/07/2024] [Accepted: 10/07/2024] [Indexed: 10/15/2024]
Abstract
Keratoconus is a common pediatric corneal disease, leading to vision impairment and amblyopia. Compared to its adult counterpart, pediatric keratoconus has an advanced presentation, rapid progression, higher incidence of complications such as corneal hydrops, and greater potential impact on the quality of life. It typically manifests during puberty and can evolve rapidly to more severe stages if left untreated. This rapid progression underscores the importance of early diagnosis through regular screening in pediatric populations and vigilant monitoring of pediatric keratoconus suspects. Concomitant ocular allergies, ocular anomalies, systemic diseases (e.g. syndromes), and poor compliance with contact lenses might impede prompt intervention and frequently postpone rehabilitation. Corneal collagen crosslinking is a crucial intervention in the management of pediatric keratoconus because it strengthens the corneal microstructure and halts the disease progression. When conservative measures fail, keratoplasty remains a viable option with generally favorable outcomes, though with unique challenges in post-operative care, including concerns related to sutures, long-term graft survival and need for repeated examinations under anesthesia. A multidisciplinary approach involving ophthalmologists, optometrists, pediatricians, and other healthcare professionals, focusing on early diagnosis and timely intervention, is essential for the comprehensive management of pediatric keratoconus and to mitigate its impact on children's lives.
Collapse
Affiliation(s)
- Yogita Gupta
- Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Delhi, India
| | - Chandradevi Shanmugam
- Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Delhi, India
| | - Priyadarshini K
- Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Delhi, India
| | - Sohini Mandal
- Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Delhi, India; LV Prasad Eye Institute, Bhubaneswar, Odisha, India
| | - Radhika Tandon
- Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Delhi, India
| | - Namrata Sharma
- Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, Delhi, India.
| |
Collapse
|
|
2
|
Sugihara K, Kaidzu S, Sasaki M, Tanito M. Interventional human ocular safety experiments for 222-nm far-ultraviolet-C lamp irradiation. Photochem Photobiol 2025; 101:517-526. [PMID: 39161063 PMCID: PMC11913762 DOI: 10.1111/php.14016] [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: 06/16/2024] [Revised: 08/01/2024] [Accepted: 08/09/2024] [Indexed: 08/21/2024]
Abstract
The study aimed to directly assess the ocular safety of 222-nm far-ultraviolet-C (UVC) irradiation in humans, given the limited clinical trials in this area. This wavelength offers the potential for safe and effective microbial inactivation in occupied spaces, but its safety profile for human eyes requires thorough investigation. This prospective, interventional study involved five subjects aged 29-47 years, who were exposed to 222-nm UVC at doses of 22, 50, and 75 mJ/cm2. The subjects were monitored using custom-made glasses with a UV-cut filter on one eye to serve as a control. UVC irradiation was conducted using a KrCl excimer lamp, and ocular examinations were performed prior to exposure, 24 h post-exposure, and at 1, 3, and 6 months. Parameters assessed included visual acuity, refractive error, corneal endothelial density, corneal erosion scores, and conjunctival hyperemia scores. The study found no clinically significant photokeratitis or long-term eye damage across the five subjects, even at the highest dose of 75 mJ/cm2. Temporary ocular discomfort, including sensations of dryness and epiphora, was reported, but these symptoms subsided within hours after irradiation. The findings indicate that 222-nm far-UVC irradiation up to 75 mJ/cm2 does not cause "clinically significant photokeratitis" or long-term ocular damage, though it may induce temporary discomfort. This supports the safe use of 222-nm UVC for germicidal applications in occupied environments, providing a basis for revised safety guidelines.
Collapse
Affiliation(s)
- Kazunobu Sugihara
- Department of OphthalmologyShimane University Faculty of MedicineIzumoJapan
| | - Sachiko Kaidzu
- Department of OphthalmologyShimane University Faculty of MedicineIzumoJapan
| | | | - Masaki Tanito
- Department of OphthalmologyShimane University Faculty of MedicineIzumoJapan
| |
Collapse
|
|
3
|
Dutra BAL, Hammoud B, Schumacher JS, Susanna BN, Asroui L, Tarib I, Sampaio LP, Scarcelli G, Roberts CJ, Randleman JB. Determining the Relationship Between Regional Epithelial Thickness, Corneal Toricity, and Corneal Power in Normal Corneas. J Refract Surg 2025; 41:e91-e101. [PMID: 39937985 DOI: 10.3928/1081597x-20250103-01] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/14/2025]
Abstract
PURPOSE To determine the relationship between regional epithelial thickness and corneal toricity and estimate the epithelium's impact on corneal power in normal corneas. METHODS This was a retrospective case-control study evaluating 200 eyes from 200 patients categorized into one of three groups based on corneal toricity obtained with Scheimpflug imaging: (1) 80 non-toric (NT) eyes (< 0.50 diopters [D] in any meridian), (2) 80 with-the-rule (WTR) eyes (⩾ +1.50 D @90 ± 22.5°), and (3) 40 against-the-rule (ATR) eyes (⩾ +1.50 D @180 ± 22.5°). Epithelial thickness maps (9 mm) were generated using anterior segment optical coherence tomography (ASOCT) imaging (Avanti RTVue XR; Optovue, Inc). Mathematical modeling was used to evaluate the epithelium's impact on corneal power. RESULTS There were no differences in mean curvature between groups (range: 43.2 to 43.7 µm). Average epithelial thickness profile within the central 9-mm zone varied by approximately 2 µm or less laterally but was thinner superiorly than inferiorly in all three groups (52.45 to 53.36 vs 56.01 to 56.48 µm) with no significant differences between groups. There were no differences in any measured epithelial metric at any location nor any deviation in overall epithelial thickness pattern between groups. There were minimal average variations (< 4 µm) across the cornea between central and peripheral values in any meridian. The modeled impact on corneal optics was a net reduction in curvature by approximately 0.30 D with less than 0.10 D of possible variation resulting from differences in regional thickness. CONCLUSIONS Regional epithelial thickness was unrelated to underlying corneal toricity and had minimal impact on corneal power in normal eyes. There was no evidence that normal epithelium significantly masks underlying normal stromal toricity, nor does the epithelium significantly contribute to corneal power under normal conditions. [J Refract Surg. 2025;41(2):e91-e101.].
Collapse
|
|
4
|
Alrashidi SH. The Corneal Epithelial Thickness Profile in a Healthy Saudi Population. Cureus 2024; 16:e71135. [PMID: 39525125 PMCID: PMC11545769 DOI: 10.7759/cureus.71135] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/09/2024] [Indexed: 11/16/2024] Open
Abstract
INTRODUCTION Epithelial mapping appears to be a valuable technique for a corneal and refractive surgeon, useful for distinguishing corneas with the true presence of corneal ectasia from those that are suspicious. Interpreting the epithelial thickness map data requires an understanding of corneal epithelial thickness (CET) normal values and variable patterns. Unlike corneal thickness, epithelial thickness assessment with anterior segment optical coherence tomography (AS-OCT) seems to be influenced by gender and age. The study aimed to investigate the detailed mapping of CET characteristics in normal eyes from the Saudi population and to assess its variation with age and sex using anterior segment 7-mm-wide OCT (AS-OCT) scans. METHODS Regional epithelial thickness was assessed using an anterior radial scanning protocol with REVO NX (Optopol Technology S.A, Zawiercie, Poland) in 596 eyes of 298 individuals aged 10 to 98. CET maps in a 7 mm diameter were automatically generated by the built-in software, displaying thickness in 17 sectors divided into three zones, i) a central zone within the 0-2 mm diameter, ii) ring 1 zone, a paracentral (P-CET) zone from 2 to 5-mm, and iii) ring 2 zone, a midperipheral zone (MP-CET) from 5 to 7 mm. Ring 1 and 2 zones were further divided into eight sectors each, including superior (S), inferior (I), nasal (N), temporal (T), superonasal (SN), inferonasal (IN), superotemporal (ST), and inferotemporal (IT). An analysis was done on correlations between age and gender and the CET across different zones. RESULTS Males and older adults had a substantially thicker CET than females and younger participants, with the C-CET measuring 59.2±4.5µm. In three zones, no interocular asymmetry was seen. Superiorly, CET is significantly thinner than inferiorly (p<0.05), with temporal zones being thinner than nasal zones (p<0.05). The C-CET increases with age in the seven groups of both genders, but its dependence on age is weaker in paracentral sectors; C-CET was 3.5% thicker in males. Paracentral nasal and inferior zones showed 2.2-3.6% thicker CET while the superior and temporal paracentral zones showed 3-5% thicker CET among males compared to females. CONCLUSION From 17 CET zones of central 7 mm cornea the C-CET was affected by gender and age. The CET distribution in these healthy Saudis' eyes was non-uniform with the CET being thinner in the superior cornea. This finding could aid in predicting corneal diseases and planning refractive procedures.
Collapse
Affiliation(s)
- Sultan H Alrashidi
- Department of Ophthalmology, College of Medicine, Qassim University, Buraidah, SAU
| |
Collapse
|
|
5
|
Verma S, Lin X, Coulson-Thomas VJ. The Potential Reversible Transition between Stem Cells and Transient-Amplifying Cells: The Limbal Epithelial Stem Cell Perspective. Cells 2024; 13:748. [PMID: 38727284 PMCID: PMC11083486 DOI: 10.3390/cells13090748] [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: 03/25/2024] [Revised: 04/18/2024] [Accepted: 04/24/2024] [Indexed: 05/13/2024] Open
Abstract
Stem cells (SCs) undergo asymmetric division, producing transit-amplifying cells (TACs) with increased proliferative potential that move into tissues and ultimately differentiate into a specialized cell type. Thus, TACs represent an intermediary state between stem cells and differentiated cells. In the cornea, a population of stem cells resides in the limbal region, named the limbal epithelial stem cells (LESCs). As LESCs proliferate, they generate TACs that move centripetally into the cornea and differentiate into corneal epithelial cells. Upon limbal injury, research suggests a population of progenitor-like cells that exists within the cornea can move centrifugally into the limbus, where they dedifferentiate into LESCs. Herein, we summarize recent advances made in understanding the mechanism that governs the differentiation of LESCs into TACs, and thereafter, into corneal epithelial cells. We also outline the evidence in support of the existence of progenitor-like cells in the cornea and whether TACs could represent a population of cells with progenitor-like capabilities within the cornea. Furthermore, to gain further insights into the dynamics of TACs in the cornea, we outline the most recent findings in other organ systems that support the hypothesis that TACs can dedifferentiate into SCs.
Collapse
Affiliation(s)
- Sudhir Verma
- College of Optometry, University of Houston, 4901 Calhoun Road, Houston, TX 77204, USA;
- Deen Dayal Upadhyaya College, University of Delhi, Delhi 110078, India
| | - Xiao Lin
- College of Optometry, University of Houston, 4901 Calhoun Road, Houston, TX 77204, USA;
| | | |
Collapse
|
|
6
|
Pan J, Pany S, Martinez-Carrasco R, Fini ME. Differential Efficacy of Small Molecules Dynasore and Mdivi-1 for the Treatment of Dry Eye Epitheliopathy or as a Countermeasure for Nitrogen Mustard Exposure of the Ocular Surface. J Pharmacol Exp Ther 2024; 388:506-517. [PMID: 37442618 PMCID: PMC10801785 DOI: 10.1124/jpet.123.001697] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2023] [Revised: 05/19/2023] [Accepted: 06/05/2023] [Indexed: 07/15/2023] Open
Abstract
The ocular surface comprises the wet mucosal epithelia of the cornea and conjunctiva, the associated glands, and the overlying tear film. Epitheliopathy is the common pathologic outcome when the ocular surface is subjected to oxidative stress. Whether different stresses act via the same or different mechanisms is not known. Dynasore and dyngo-4a, small molecules developed to inhibit the GTPase activity of classic dynamins DNM1, DNM2, and DNM3, but not mdivi-1, a specific inhibitor of DNM1L, protect corneal epithelial cells exposed to the oxidant tert-butyl hydroperoxide (tBHP). Here we report that, while dyngo-4a is the more potent inhibitor of endocytosis, dynasore is the better cytoprotectant. Dynasore also protects corneal epithelial cells against exposure to high salt in an in vitro model of dysfunctional tears in dry eye. We now validate this finding in vivo, demonstrating that dynasore protects against epitheliopathy in a mouse model of dry eye. Knockdown of classic dynamin DNM2 was also cytoprotective against tBHP exposure, suggesting that dynasore's effect is at least partially on target. Like tBHP and high salt, exposure of corneal epithelial cells to nitrogen mustard upregulated the unfolded protein response and inflammatory markers, but dynasore did not protect against nitrogen mustard exposure. In contrast, mdivi-1 was cytoprotective. Interestingly, mdivi-1 did not inhibit the nitrogen mustard-induced expression of inflammatory cytokines. We conclude that exposure to tBHP or nitrogen mustard, two different oxidative stress agents, cause corneal epitheliopathy via different pathologic pathways. SIGNIFICANCE STATEMENT: Results presented in this paper, for the first time, implicate the dynamin DNM2 in ocular surface epitheliopathy. The findings suggest that dynasore could serve as a new topical treatment for dry eye epitheliopathy and that mdivi-1 could serve as a medical countermeasure for epitheliopathy due to nitrogen mustard exposure, with potentially increased efficacy when combined with anti-inflammatory agents and/or UPR modulators.
Collapse
Affiliation(s)
- Jinhong Pan
- New England Eye Center, Tufts Medical Center and Department of Ophthalmology, Tufts University School of Medicine (J.P., S.P., R.M.-C., M.E.F.) and Program in Pharmacology and Drug Development, Tufts Graduate School of Biomedical Sciences (M.E.F.), Tufts University, Boston, Massachusetts
| | - Satyabrata Pany
- New England Eye Center, Tufts Medical Center and Department of Ophthalmology, Tufts University School of Medicine (J.P., S.P., R.M.-C., M.E.F.) and Program in Pharmacology and Drug Development, Tufts Graduate School of Biomedical Sciences (M.E.F.), Tufts University, Boston, Massachusetts
| | - Rafael Martinez-Carrasco
- New England Eye Center, Tufts Medical Center and Department of Ophthalmology, Tufts University School of Medicine (J.P., S.P., R.M.-C., M.E.F.) and Program in Pharmacology and Drug Development, Tufts Graduate School of Biomedical Sciences (M.E.F.), Tufts University, Boston, Massachusetts
| | - M Elizabeth Fini
- New England Eye Center, Tufts Medical Center and Department of Ophthalmology, Tufts University School of Medicine (J.P., S.P., R.M.-C., M.E.F.) and Program in Pharmacology and Drug Development, Tufts Graduate School of Biomedical Sciences (M.E.F.), Tufts University, Boston, Massachusetts
| |
Collapse
|
|
7
|
Azzari NA, Segars KL, Rapaka S, Kushimi L, Rich CB, Trinkaus-Randall V. Aberrations in Cell Signaling Quantified in Diabetic Murine Globes after Injury. Cells 2023; 13:26. [PMID: 38201230 PMCID: PMC10778404 DOI: 10.3390/cells13010026] [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: 11/01/2023] [Revised: 12/12/2023] [Accepted: 12/19/2023] [Indexed: 01/12/2024] Open
Abstract
The corneal epithelium is an avascular structure that has a unique wound healing mechanism, which allows for rapid wound closure without compromising vision. This wound healing mechanism is attenuated in diabetic patients, resulting in poor clinical outcomes and recurrent non-healing erosion. We investigated changes in cellular calcium signaling activity during the wound response in murine diabetic tissue using live cell imaging from both ex vivo and in vitro models. The calcium signaling propagation in diabetic cells was significantly decreased and displayed altered patterns compared to non-diabetic controls. Diabetic cells and tissue display distinct expression of the purinergic receptor, P2X7, which mediates the wound healing response. We speculate that alterations in P2X7 expression, interactions with other proteins, and calcium signaling activity significantly impact the wound healing response. This may explain aberrations in the diabetic wound response.
Collapse
Affiliation(s)
- Nicholas A. Azzari
- Department of Biochemistry and Cell Biology, Boston University Chobanian and Avedisian School of Medicine, 72 E. Concord St., Boston, MA 02118, USA; (N.A.A.); (C.B.R.)
| | - Kristen L. Segars
- Department of Pharmacology, Physiology and Biophysics, Boston University Chobanian and Avedisian School of Medicine, 72 E. Concord St., Boston, MA 02118, USA;
| | - Srikar Rapaka
- Department of Medicine, Boston University Chobanian and Avedisian School of Medicine, 72 E. Concord St., Boston, MA 02118, USA;
| | - Landon Kushimi
- Department of Computer Science, Center for Computing and Data Sciences, Boston University, 665 Commonwealth Ave, Boston, MA 02115, USA;
| | - Celeste B. Rich
- Department of Biochemistry and Cell Biology, Boston University Chobanian and Avedisian School of Medicine, 72 E. Concord St., Boston, MA 02118, USA; (N.A.A.); (C.B.R.)
| | - Vickery Trinkaus-Randall
- Department of Biochemistry and Cell Biology, Boston University Chobanian and Avedisian School of Medicine, 72 E. Concord St., Boston, MA 02118, USA; (N.A.A.); (C.B.R.)
- Department of Pharmacology, Physiology and Biophysics, Boston University Chobanian and Avedisian School of Medicine, 72 E. Concord St., Boston, MA 02118, USA;
- Department of Ophthalmology, Boston University Chobanian and Avedisian School of Medicine, 72 E. Concord St., Boston, MA 02118, USA
| |
Collapse
|
|
8
|
Vottonen L, Koskela A, Felszeghy S, Wylegala A, Kryszan K, Gurubaran IS, Kaarniranta K, Wylegala E. Oxidative Stress and Cellular Protein Accumulation Are Present in Keratoconus, Macular Corneal Dystrophy, and Fuchs Endothelial Corneal Dystrophy. J Clin Med 2023; 12:4332. [PMID: 37445366 DOI: 10.3390/jcm12134332] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2023] [Revised: 06/16/2023] [Accepted: 06/26/2023] [Indexed: 07/15/2023] Open
Abstract
The aim of the study was to investigate oxidative stress as well as cellular protein accumulation in corneal diseases including keratoconus (KC), macular corneal dystrophy (MCD), and Fuchs endothelial corneal dystrophy (FECD) at their primary affecting sites. Corneal buttons from KC, MCD, and FECD patients, as well as healthy controls, were analyzed immunohistochemically to evaluate the presence of oxidative stress and the function of the proteostasis network. 4-Fydroxynonenal (4-HNE) was used as a marker of oxidative stress, whereas the levels of catalase and heat-shock protein 70 (HSP70) were analyzed to evaluate the response of the antioxidant defense system and molecular chaperones, respectively. Sequestosome 1 (SQSTM1) levels were determined to assess protein aggregation and the functionality of autophagic degradation. Basal epithelial cells of the KC samples showed increased levels of oxidative stress marker 4-HNE and antioxidant enzyme catalase together with elevated levels of HSP70 and accumulation of SQSTM1. Corneal stromal cells and endothelial cells from MCD and FECD samples, respectively, showed similarly increased levels of these markers. All corneal diseases showed the presence of oxidative stress and activation of the molecular chaperone response to sustain protein homeostasis. However, the accumulation of protein aggregates suggests insufficient function of the protective mechanisms to limit the oxidative damage and removal of protein aggregates via autophagy. These results suggest that oxidative stress has a role in KC, MCD, and FECD at the cellular level as a secondary outcome. Thus, antioxidant- and autophagy-targeted therapies could be included as supporting care when treating KC or corneal dystrophies.
Collapse
Affiliation(s)
- Linda Vottonen
- Department of Ophthalmology, Kuopio University Hospital, 70210 Kuopio, Finland
| | - Ali Koskela
- Department of Ophthalmology, University of Eastern Finland, 70210 Kuopio, Finland
| | - Szabolcs Felszeghy
- Institute of Biomedicine, University of Eastern Finland, Yliopistonranta 1, 70210 Kuopio, Finland
| | - Adam Wylegala
- Health Promotion and Obesity Management Unit, Department of Pathophysiology, Faculty of Medical Sciences, Medical University of Silesia, 40-055 Katowice, Poland
- Ophthalmology Department, Railway Hospital, 40-760 Katowice, Poland
| | | | | | - Kai Kaarniranta
- Department of Ophthalmology, Kuopio University Hospital, 70210 Kuopio, Finland
- Department of Ophthalmology, University of Eastern Finland, 70210 Kuopio, Finland
- Department of Molecular Genetics, Faculty of Biology and Environmental Protection, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland
| | - Edward Wylegala
- Ophthalmology Department, Railway Hospital, 40-760 Katowice, Poland
- Clinical Department of Ophthalmology, II School of Medicine with the Division of Dentistry in Zabrze, Medical University of Silesia, 40-760 Katowice, Poland
| |
Collapse
|
|
9
|
Martinez-Carrasco R, Fini ME. Dynasore Protects Corneal Epithelial Cells Subjected to Hyperosmolar Stress in an In Vitro Model of Dry Eye Epitheliopathy. Int J Mol Sci 2023; 24:ijms24054754. [PMID: 36902183 PMCID: PMC10003680 DOI: 10.3390/ijms24054754] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 02/19/2023] [Accepted: 02/20/2023] [Indexed: 03/06/2023] Open
Abstract
Epitheliopathy at the ocular surface is a defining sign of dry eye disease, a common disorder that affects 10% to 30% of the world's population. Hyperosmolarity of the tear film is one of the main drivers of pathology, with subsequent endoplasmic reticulum (ER) stress, the resulting unfolded protein response (UPR), and caspase-3 activation implicated in the pathway to programmed cell death. Dynasore, is a small molecule inhibitor of dynamin GTPases that has shown therapeutic effects in a variety of disease models involving oxidative stress. Recently we showed that dynasore protects corneal epithelial cells exposed to the oxidant tBHP, by selective reduction in expression of CHOP, a marker of the UPR PERK branch. Here we investigated the capacity of dynasore to protect corneal epithelial cells subjected to hyperosmotic stress (HOS). Similar to dynasore's capacity to protect against tBHP exposure, dynasore inhibits the cell death pathway triggered by HOS, protecting against ER stress and maintaining a homeostatic level of UPR activity. However, unlike with tBHP exposure, UPR activation due to HOS is independent of PERK and mostly driven by the UPR IRE1 branch. Our results demonstrate the role of the UPR in HOS-driven damage, and the potential of dynasore as a treatment to prevent dry eye epitheliopathy.
Collapse
Affiliation(s)
- Rafael Martinez-Carrasco
- New England Eye Center, Tufts Medical Center and Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02111, USA
- Correspondence: (R.M.-C.); (M.E.F.)
| | - M. Elizabeth Fini
- New England Eye Center, Tufts Medical Center and Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02111, USA
- Graduate School of Biomedical Sciences, Tufts University, Boston, MA 02111, USA
- Correspondence: (R.M.-C.); (M.E.F.)
| |
Collapse
|
|
10
|
Liton PB, Boesze-Battaglia K, Boulton ME, Boya P, Ferguson TA, Ganley IG, Kauppinnen A, Laurie GW, Mizushima N, Morishita H, Russo R, Sadda J, Shyam R, Sinha D, Thompson DA, Zacks DN. AUTOPHAGY IN THE EYE: FROM PHYSIOLOGY TO PATHOPHYSOLOGY. AUTOPHAGY REPORTS 2023; 2:2178996. [PMID: 37034386 PMCID: PMC10078619 DOI: 10.1080/27694127.2023.2178996] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/19/2022] [Accepted: 01/26/2023] [Indexed: 03/05/2023]
Abstract
Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye.
Collapse
Affiliation(s)
- Paloma B. Liton
- Departments of Ophthalmology & Pathology, Duke School of Medicine, Duke University, Durham, NC 27705, USA
| | - Kathleen Boesze-Battaglia
- Department of Basic and Translational Sciences, University of Pennsylvania, School of Dental Medicine, Philadelphia, PA 19104, USA
| | - Michael E. Boulton
- Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham (UAB), Birmingham, AL, USA
| | - Patricia Boya
- Department of Neuroscience and Movement Science. Faculty of Science and Medicine, University of Fribourg, 1700 Fribourg, Switzerland
| | - Thomas A. Ferguson
- Department of Ophthalmology and Visual Sciences, Washington University in St. Louis, St. Louis, MO 63110, USA
| | - Ian G. Ganley
- MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK
| | - Anu Kauppinnen
- Faculty of Health and Sciences, School of Pharmacy, University of Eastern Finland, 70210 Kuopio, Finland
| | - Gordon W. Laurie
- Departments of Cell Biology, Ophthalmology and Biomedical Engineering, University of Virginia, Charlottesville, VA 22908, USA
| | - Noboru Mizushima
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, 113-0033, Japan
| | - Hideaki Morishita
- Department of Biochemistry and Molecular Biology, Graduate School of Medicine, The University of Tokyo, 113-0033, Japan
- Department of Physiology, Juntendo University Graduate School of Medicine, Bunkyo-ku, Tokyo, 113-8421, Japan
| | - Rossella Russo
- Preclinical and Translational Pharmacology, Glaucoma Unit, Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Rende, Italy
| | - Jaya Sadda
- Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan Medical School, Ann Arbor, MI, USA
| | | | - Debasish Sinha
- Department of Ophthalmology, Cell Biology, and Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
- Wilmer Eye Institute, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Debra A. Thompson
- Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan Medical School, Ann Arbor, MI, USA
| | - David N. Zacks
- Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, University of Michigan Medical School, Ann Arbor, MI, USA
| |
Collapse
|
|
11
|
Chintala SK, Pan J, Satapathy S, Condruti R, Hao Z, Liu PW, O’Conner CF, Barr JT, Wilson MR, Jeong S, Fini ME. Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy. Int J Mol Sci 2023; 24:981. [PMID: 36674497 PMCID: PMC9861099 DOI: 10.3390/ijms24020981] [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: 11/21/2022] [Revised: 12/23/2022] [Accepted: 12/31/2022] [Indexed: 01/07/2023] Open
Abstract
There is a significant unmet need for therapeutics to treat ocular surface barrier damage, also called epitheliopathy, due to dry eye and related diseases. We recently reported that the natural tear glycoprotein CLU (clusterin), a molecular chaperone and matrix metalloproteinase inhibitor, seals and heals epitheliopathy in mice subjected to desiccating stress in a model of aqueous-deficient/evaporative dry eye. Here we investigated CLU sealing using a second model with features of ophthalmic preservative-induced dry eye. The ocular surface was stressed by topical application of the ophthalmic preservative benzalkonium chloride (BAC). Then eyes were treated with CLU and sealing was evaluated immediately by quantification of clinical dye uptake. A commercial recombinant form of human CLU (rhCLU), as well as an rhCLU form produced in our laboratory, designed to be compatible with U.S. Food and Drug Administration guidelines on current Good Manufacturing Practices (cGMP), were as effective as natural plasma-derived human CLU (pCLU) in sealing the damaged ocular surface barrier. In contrast, two other proteins found in tears: TIMP1 and LCN1 (tear lipocalin), exhibited no sealing activity. The efficacy and selectivity of rhCLU for sealing of the damaged ocular surface epithelial barrier suggests that it could be of therapeutic value in treating BAC-induced epitheliopathy and related diseases.
Collapse
Affiliation(s)
- Shravan K. Chintala
- USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA 90033, USA
| | - Jinhong Pan
- New England Eye Center, Tufts Medical Center, Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02111, USA
| | - Sandeep Satapathy
- School of Chemistry and Molecular Bioscience, Molecular Horizons Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia
| | - Rebecca Condruti
- Training Program in Cell, Molecular and Developmental Biology, Graduate School of Biomedical Sciences, Tufts University, Boston, MA 02111, USA
| | - Zixuan Hao
- Training Program in Pharmacology and Drug Development, Graduate School of Biomedical Sciences, Tufts University, Boston, MA 02111, USA
| | - Pei-wen Liu
- Training Program in Pharmacology and Drug Development, Graduate School of Biomedical Sciences, Tufts University, Boston, MA 02111, USA
| | - Christian F. O’Conner
- Doctor of Medicine Training Program, Tufts University School of Medicine, Boston, MA 02111, USA
| | - Joseph T. Barr
- The Ohio State University College of Optometry, Columbus, OH 43210, USA
| | - Mark R. Wilson
- School of Chemistry and Molecular Bioscience, Molecular Horizons Research Institute, University of Wollongong, Wollongong, NSW 2522, Australia
| | - Shinwu Jeong
- USC Roski Eye Institute, Department of Ophthalmology, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA 90033, USA
| | - M. Elizabeth Fini
- New England Eye Center, Tufts Medical Center, Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02111, USA
| |
Collapse
|
|
12
|
In situ transduction of cells in human corneal limbus using adeno-associated viruses: an ex vivo study. Sci Rep 2022; 12:22481. [PMID: 36577775 PMCID: PMC9797548 DOI: 10.1038/s41598-022-26926-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2022] [Accepted: 12/21/2022] [Indexed: 12/30/2022] Open
Abstract
This study aimed to evaluate the efficacy of in situ adeno-associated virus (AAV)-mediated gene delivery into the human corneal limbal region via targeted sub-limbal injection technique. Human cadaveric corneal tissues were fixed on an artificial anterior chamber. Feasibility of sub-limbal injection technique was tested using trypan blue and black India ink. An enhanced green fluorescent protein (eGFP) encoding AAV DJ was injected into sub-limbal region. After AAV injection, corneal tissues were incubated in air-lift culture and prepared for immunohistochemical analysis. Cell survivial and expression of eGFP, stem cell markers (p63α and cytokeratin 19 (KRT19)), and differentiation marker cytokeratin 3 (KRT3) were evaluated using confocal microscopy. Both trypan blue and black India ink stained and were retained sub-limbally establishing specificity of the injection technique. Immunohistochemical analysis of corneas injected with AAV DJ-eGFP indicated that AAV-transduced cells in the limbal region co-express eGFP, p63α, and KRT19 and that these transduced cells were capable of differentiating to KRT3 postitive corneal epithelial cells. Our sub-limbal injection technique can target cells in the human limbus in a reproducible and efficient manner. Thus, we demonstrate that in situ injection of corneal limbus may provide a feasible mode of genetic therapy for corneal disorders with an epithelial etiology.
Collapse
|
|
13
|
Sklenářová R, Akla N, Latorre MJ, Ulrichová J, Franková J. Collagen as a Biomaterial for Skin and Corneal Wound Healing. J Funct Biomater 2022; 13:jfb13040249. [PMID: 36412890 PMCID: PMC9680244 DOI: 10.3390/jfb13040249] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2022] [Revised: 11/11/2022] [Accepted: 11/15/2022] [Indexed: 11/18/2022] Open
Abstract
The cornea and the skin are two organs that form the outer barrier of the human body. When either is injured (e.g., from surgery, physical trauma, or chemical burns), wound healing is initiated to restore integrity. Many cells are activated during wound healing. In particular, fibroblasts that are stimulated often transition into repair fibroblasts or myofibroblasts that synthesize extracellular matrix (ECM) components into the wound area. Control of wound ECM deposition is critical, as a disorganized ECM can block restoration of function. One of the most abundant structural proteins in the mammalian ECM is collagen. Collagen type I is the main component in connective tissues. It can be readily obtained and purified, and short analogs have also been developed for tissue engineering applications, including modulating the wound healing response. This review discusses the effect of several current collagen implants on the stimulation of corneal and skin wound healing. These range from collagen sponges and hydrogels to films and membranes.
Collapse
Affiliation(s)
- Renáta Sklenářová
- Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacký University in Olomouc, 775 15 Olomouc, Czech Republic
- Maisonneuve-Rosemont Hospital Research Centre, Montréal, QC H1T 2M4, Canada
| | - Naoufal Akla
- Maisonneuve-Rosemont Hospital Research Centre, Montréal, QC H1T 2M4, Canada
- Department of Ophthalmology, Université de Montréal, Montréal, QC H3C 3J7, Canada
| | | | - Jitka Ulrichová
- Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacký University in Olomouc, 775 15 Olomouc, Czech Republic
| | - Jana Franková
- Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacký University in Olomouc, 775 15 Olomouc, Czech Republic
- Correspondence:
| |
Collapse
|
|
14
|
Reinstein DZ, Archer TJ, Vida RS. Epithelial thickness mapping for corneal refractive surgery. Curr Opin Ophthalmol 2022; 33:258-268. [PMID: 35779050 DOI: 10.1097/icu.0000000000000867] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
PURPOSE OF REVIEW As more devices become available that offer corneal epithelial thickness mapping, this is becoming more widely used for numerous applications in corneal refractive surgery. RECENT FINDINGS The epithelial thickness profile is nonuniform in the normal eye, being thinner superiorly than inferiorly and thinner temporally than nasally. Changes in the epithelial thickness profile are highly predictable, responding to compensate for changes in the stromal curvature gradient, using the eyelid as an outer template. This leads to characteristic changes that can be used for early screening in keratoconus, postoperative monitoring for early signs of corneal ectasia, and for determining whether further steepening can be performed without the risk of apical syndrome following primary hyperopic treatment. Compensatory epithelial thickness changes are also a critical part of diagnosis in irregular astigmatism as these partially mask the stromal surface irregularities. The epithelial thickness map can then be used to plan a trans-epithelial PRK treatment for cases of irregularly irregular astigmatism. Other factors can also affect the epithelial thickness profile, including dry eye, anterior basement membrane dystrophy and eyelid ptosis. SUMMARY Epithelial thickness mapping is becoming a crucial tool for refractive surgery, in particular for keratoconus screening, ectasia monitoring, hyperopic treatment planning, and therapeutic diagnosis and treatment.
Collapse
Affiliation(s)
- Dan Z Reinstein
- Reinstein Vision
- London Vision Clinic, London, UK
- Department of Ophthalmology, Columbia University Medical Center, New York, USA
- Sorbonne Université, Paris, France
- School of Biomedical Sciences, University of Ulster, Coleraine, UK
| | | | | |
Collapse
|
|
15
|
O'leary DJ, Wilson G. Tear‐side re̊gulation of desquamation in the rabbit corneal epithelium: A specular microscopy study. Clin Exp Optom 2021. [DOI: 10.1111/j.1444-0938.1986.tb04562.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Affiliation(s)
- Daniel J. O'leary
- Laboratory of Experimental Optometry, University of Wales Institute of Science and Technology
| | - Graham Wilson
- Department of Physiological Optics, School of Optometry, The University of Alabama in Birmingham
| |
Collapse
|
|
16
|
Efron N. Putting vital stains in context. Clin Exp Optom 2021; 96:400-21. [DOI: 10.1111/j.1444-0938.2012.00802.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2011] [Revised: 06/17/2012] [Accepted: 06/19/2012] [Indexed: 11/30/2022] Open
Affiliation(s)
- Nathan Efron
- Institute of Health and Biomedical Innovation, and School of Optometry and Vision Science, Queensland University of Technology, Kelvin Grove, Queensland, Australia,
| |
Collapse
|
|
17
|
Adil MT, Henry JJ. Understanding cornea epithelial stem cells and stem cell deficiency: Lessons learned using vertebrate model systems. Genesis 2021; 59:e23411. [PMID: 33576188 DOI: 10.1002/dvg.23411] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2020] [Revised: 01/08/2021] [Accepted: 01/09/2021] [Indexed: 12/13/2022]
Abstract
Animal models have contributed greatly to our understanding of human diseases. Here, we focus on cornea epithelial stem cell (CESC) deficiency (commonly called limbal stem cell deficiency, LSCD). Corneal development, homeostasis and wound healing are supported by specific stem cells, that include the CESCs. Damage to or loss of these cells results in blindness and other debilitating ocular conditions. Here we describe the contributions from several vertebrate models toward understanding CESCs and LSCD treatments. These include both mammalian models, as well as two aquatic models, Zebrafish and the amphibian, Xenopus. Pioneering developments have been made using stem cell transplants to restore normal vision in patients with LSCD, but questions still remain about the basic biology of CESCs, including their precise cell lineages and behavior in the cornea. We describe various cell lineage tracing studies to follow their patterns of division, and the fates of their progeny during development, homeostasis, and wound healing. In addition, we present some preliminary results using the Xenopus model system. Ultimately, a more thorough understanding of these cornea cells will advance our knowledge of stem cell biology and lead to better cornea disease therapeutics.
Collapse
Affiliation(s)
- Mohd Tayyab Adil
- Department of Cell & Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
| | - Jonathan J Henry
- Department of Cell & Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
| |
Collapse
|
|
18
|
Fini ME, Jeong S, Wilson MR. Therapeutic Potential of the Molecular Chaperone and Matrix Metalloproteinase Inhibitor Clusterin for Dry Eye. Int J Mol Sci 2020; 22:E116. [PMID: 33374364 PMCID: PMC7794831 DOI: 10.3390/ijms22010116] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Revised: 12/19/2020] [Accepted: 12/21/2020] [Indexed: 12/29/2022] Open
Abstract
Evidence is presented herein supporting the potential of the natural homeostatic glycoprotein CLU (clusterin) as a novel therapeutic for the treatment of dry eye. This idea began with the demonstration that matrix metalloproteinase MMP9 is required for damage to the ocular surface in mouse dry eye. Damage was characterized by degradation of OCLN (occludin), a known substrate of MMP9 and a key component of the paracellular barrier. Following up on this finding, a yeast two-hybrid screen was conducted using MMP9 as the bait to identify other proteins involved. CLU emerged as a strong interacting protein that inhibits the enzymatic activity of MMP9. Previously characterized as a molecular chaperone, CLU is expressed prominently by epithelia at fluid-tissue interfaces and secreted into bodily fluids, where it protects cells and tissues against damaging stress. It was demonstrated that CLU also protects the ocular surface in mouse dry eye when applied topically to replace the natural protein depleted from the dysfunctional tears. CLU is similarly depleted from tears in human dry eye. The most novel and interesting finding was that CLU binds selectively to the damaged ocular surface. In this position, CLU protects against epithelial cell death and barrier proteolysis, and dampens the autoimmune response, while the apical epithelial cell layer is renewed. When present at high enough concentration, CLU also blocks staining by vital dyes used clinically to diagnose dry eye. None of the current therapeutics have this combination of properties to "protect, seal, and heal". Future work will be directed towards human clinical trials to investigate the therapeutic promise of CLU.
Collapse
Affiliation(s)
- M. Elizabeth Fini
- New England Eye Center, Tufts Medical Center and Department of Ophthalmology, Tufts University School of Medicine, Program in Pharmacology & Drug Development, Graduate School of Biomedical Sciences Tufts University, Boston, MA 02111, USA
| | - Shinwu Jeong
- Department of Ophthalmology, USC Roski Eye Institute, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA 90089, USA;
| | - Mark R. Wilson
- The Illawarra Health and Medical Research Institute, Molecular Horizons and the School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW 2522, Australia;
| |
Collapse
|
|
19
|
Collin SP, Collin HB. A comparison of the ultrastructure of the cornea of the pre- and post-metamorphic axolotl (Ambystoma mexicanum, Amphibia). Exp Eye Res 2020; 202:108396. [PMID: 33310055 DOI: 10.1016/j.exer.2020.108396] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2020] [Revised: 11/21/2020] [Accepted: 12/07/2020] [Indexed: 11/24/2022]
Abstract
The corneal ultrastructure of the pre- and post-metamorphic stages of the neotenic axolotl Ambystoma mexicanum is examined using light microscopy and both scanning and transmission electron microscopy to reveal whether there are any morphological changes associated with a switch in lifestyle. Although the complement of corneal layers remains the same, there are significant quantitative changes in corneal, epithelial and stromal thickness, epithelial and endothelial cell size and density, and the thickness of Bowman's layer and Desçemet's membrane. Microholes in the epithelium and vertical sutures within the stroma are predominant features in the pre-metamorphic stage but are rarely seen in the post-metamorphic stage. There are also significant quantitative centro-peripheral differences in the thickness of the whole cornea, primarily due to differences in the thickness of the stroma in both metamorphic stages. These changes may reflect the physiological demands on the cornea as it switches from a purely aquatic to an amphibious lifestyle, which includes venturing onto land.
Collapse
Affiliation(s)
- Shaun P Collin
- School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia; Oceans Graduate School and the Oceans Institute, The University of Western Australia, Crawley, 6009, Western Australia, Australia.
| | - H Barry Collin
- Department of Optometry and Vision Science, University of New South Wales, Kensington, 2052, New South Wales, Australia.
| |
Collapse
|
|
20
|
Seyed-Safi AG, Daniels JT. The limbus: Structure and function. Exp Eye Res 2020; 197:108074. [PMID: 32502532 DOI: 10.1016/j.exer.2020.108074] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 05/14/2020] [Accepted: 05/17/2020] [Indexed: 12/12/2022]
Abstract
Limbal function is a key determinant of corneal epithelial integrity. Lineage tracing studies in mice have highlighted that the centripetal movement of epithelial progenitors from the limbus drives both the steady-state maintenance of the corneal epithelium and its regeneration following injury. It is well established that this is facilitated by a population of limbal epithelial stem cells within the limbus. It is becoming increasingly apparent that the behaviour of these stem cells and their ability to respond to the needs of the tissue are closely linked to their immediate microenvironment - the stem cell niche. Increasing understanding of the structural features of this niche and the signalling networks that they coordinate is required to enhance the therapeutic application of these cells in the treatment of limbal stem cell deficiency. Importantly, an improved characterisation of the hierarchy of limbal epithelial progenitors using both new and old putative markers will enable a greater appreciation for the effects of many of these limbal niche factors on stem cell fate.
Collapse
|
|
21
|
Utility of regional epithelial thickness measurements in corneal evaluations. Surv Ophthalmol 2020; 65:187-204. [DOI: 10.1016/j.survophthal.2019.09.003] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Revised: 09/10/2019] [Accepted: 09/16/2019] [Indexed: 11/19/2022]
|
|
22
|
Fini ME, Jeong S, Gong H, Martinez-Carrasco R, Laver NMV, Hijikata M, Keicho N, Argüeso P. Membrane-associated mucins of the ocular surface: New genes, new protein functions and new biological roles in human and mouse. Prog Retin Eye Res 2019; 75:100777. [PMID: 31493487 DOI: 10.1016/j.preteyeres.2019.100777] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2019] [Revised: 08/26/2019] [Accepted: 08/31/2019] [Indexed: 01/17/2023]
Abstract
The mucosal glycocalyx of the ocular surface constitutes the point of interaction between the tear film and the apical epithelial cells. Membrane-associated mucins (MAMs) are the defining molecules of the glycocalyx in all mucosal epithelia. Long recognized for their biophysical properties of hydration, lubrication, anti-adhesion and repulsion, MAMs maintain the wet ocular surface, lubricate the blink, stabilize the tear film and create a physical barrier to the outside world. However, it is increasingly appreciated that MAMs also function as cell surface receptors that transduce information from the outside to the inside of the cell. A number of excellent review articles have provided perspective on the field as it has progressed since 1987, when molecular cloning of the first MAM was reported. The current article provides an update for the ocular surface, placing it into the broad context of findings made in other organ systems, and including new genes, new protein functions and new biological roles. We discuss the epithelial tissue-equivalent with mucosal differentiation, the key model system making these advances possible. In addition, we make the first systematic comparison of MAMs in human and mouse, establishing the basis for using knockout mice for investigations with the complexity of an in vivo system. Lastly, we discuss findings from human genetics/genomics, which are providing clues to new MAM roles previously unimagined. Taken together, this information allows us to generate hypotheses for the next stage of investigation to expand our knowledge of MAM function in intracellular signaling and roles unique to the ocular surface.
Collapse
Affiliation(s)
- M Elizabeth Fini
- Department of Ophthalmology, Tufts University School of Medicine, at New England Eye Center, Tufts Medical Center, 800 Washington St, Boston, MA, 02111, USA.
| | - Shinwu Jeong
- USC Roski Eye Institute and Department of Ophthalmology, Keck School of Medicine of USC, University of Southern California, 1975 Zonal Ave, Los Angeles, CA, 90033, USA.
| | - Haiyan Gong
- Department of Ophthalmology, Boston University School of Medicine, 72 E Concord St, Boston, MA, 02118, USA.
| | - Rafael Martinez-Carrasco
- Department of Ophthalmology, Tufts University School of Medicine, at New England Eye Center, Tufts Medical Center, 800 Washington St, Boston, MA, 02111, USA.
| | - Nora M V Laver
- Department of Ophthalmology, Tufts University School of Medicine, at New England Eye Center, Tufts Medical Center, 800 Washington St, Boston, MA, 02111, USA.
| | - Minako Hijikata
- Department of Pathophysiology and Host Defense, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama, Kiyose-shi, Tokyo, 204-8533, Japan.
| | - Naoto Keicho
- Department of Pathophysiology and Host Defense, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama, Kiyose-shi, Tokyo, 204-8533, Japan.
| | - Pablo Argüeso
- Department of Ophthalmology, Harvard Medical School, at Schepens Eye Research Institute of Mass. Eye and Ear, 20 Staniford St, Boston, MA, 02114, USA.
| |
Collapse
|
|
23
|
Ruffin M, Brochiero E. Repair Process Impairment by Pseudomonas aeruginosa in Epithelial Tissues: Major Features and Potential Therapeutic Avenues. Front Cell Infect Microbiol 2019; 9:182. [PMID: 31214514 PMCID: PMC6554286 DOI: 10.3389/fcimb.2019.00182] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2018] [Accepted: 05/13/2019] [Indexed: 01/13/2023] Open
Abstract
Epithelial tissues protecting organs from the environment are the first-line of defense against pathogens. Therefore, efficient repair mechanisms after injury are crucial to maintain epithelial integrity. However, these healing processes can be insufficient to restore epithelial integrity, notably in infectious conditions. Pseudomonas aeruginosa infections in cutaneous, corneal, and respiratory tract epithelia are of particular concern because they are the leading causes of hospitalizations, disabilities, and deaths worldwide. Pseudomonas aeruginosa has been shown to alter repair processes, leading to chronic wounds and infections. Because of the current increase in the incidence of multi-drug resistant isolates of P. aeruginosa, complementary approaches to decrease the negative impact of these bacteria on epithelia are urgently needed. Here, we review the recent advances in the understanding of the impact of P. aeruginosa infections on the integrity and repair mechanisms of alveolar, airway, cutaneous and corneal epithelia. Potential therapeutic avenues aimed at counteracting this deleterious impact of infection are also discussed.
Collapse
Affiliation(s)
- Manon Ruffin
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada.,Département de Médecine, Université de Montréal, Montréal, QC, Canada.,INSERM, Centre de Recherche Saint-Antoine, CRSA, Sorbonne Université, Paris, France
| | - Emmanuelle Brochiero
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, QC, Canada.,Département de Médecine, Université de Montréal, Montréal, QC, Canada
| |
Collapse
|
|
24
|
Saffar Shahroodi A, Nejabat M, Nimrouzi M, Aghaei H, Salehi A, Rezaei Mokarram A. Effects of intranasal administration of violet oil in dry eye disease. Clin Exp Optom 2019; 102:576-582. [PMID: 31060104 DOI: 10.1111/cxo.12910] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2018] [Revised: 02/03/2019] [Accepted: 03/20/2019] [Indexed: 11/28/2022] Open
Abstract
BACKGROUND Dry eye disease is a disorder of the tear film. In this study, the effect of Viola odorata L. oily extract was examined for the treatment of patients suffering from dry eye disease. METHODS A randomised, double-blind, placebo-controlled study was designed. During the trial, Schirmer's test, tear breakup time, Oxford staining and the Ocular Surface Disease Index were assessed. Overall, 105 patients with dry eye symptoms between the ages of 18 and 60 years were allocated to the violet-almond oil, almond oil and placebo (1% w/v hydroxypropyl methylcellulose solution) groups. The treatment and placebo were administered intranasally, two drops three times a day for one month. The patients were followed up for four weeks. A total of 91 patients (32, 29 and 30 in the violet-almond oil, almond oil and placebo groups, respectively) completed the study. RESULTS At baseline, there was no difference between the three groups in terms of demographic data and the measurement parameters. After the intervention, the results revealed that the Schirmer's score without local anaesthesia and the tear breakup time results significantly improved in the violet-almond oil group. One-way ANOVA indicated a significant improvement in the Schirmer's score, tear breakup time and Ocular Surface Disease Index of the treatment group, as compared with the other groups (p < 0.05). However, the obtained results did not present any significant mean difference between and within the groups of the Oxford staining grade (p > 0.05). CONCLUSIONS This trial showed that the intranasally administered V. odorata L. oily extract enhances tear production and improves tear film stability.
Collapse
Affiliation(s)
- Aniseh Saffar Shahroodi
- Department of Traditional Persian Medicine, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.,Research Center for Traditional Medicine and History of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mahmood Nejabat
- Poostchi Ophthalmology Research Centre, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Majid Nimrouzi
- Department of Traditional Persian Medicine, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.,Research Center for Traditional Medicine and History of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Hossein Aghaei
- Eye Research Center, Rassoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran
| | - Alireza Salehi
- Research Center for Traditional Medicine and History of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Ali Rezaei Mokarram
- Department of Biology, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran
| |
Collapse
|
|
25
|
Effect of D-Panthenol on Corneal Epithelial Healing after Surface Laser Ablation. J Ophthalmol 2018; 2018:6537413. [PMID: 30538855 PMCID: PMC6260525 DOI: 10.1155/2018/6537413] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2018] [Accepted: 10/09/2018] [Indexed: 11/17/2022] Open
Abstract
Purpose To study the effect of D-panthenol (provitamin B5) on corneal epithelial healing, in cases of surface laser ablation. Patients and Methods: 45 eyes, of 45 patients undergoing laser surface ablation, received D-panthenol 2% in one eye and artificial tear drops of similar composition not containing D-panthenol in the other eye, postoperatively, for 2 months. Patients were examined daily for 3 days after the procedure. They were then examined weekly for 1 month. An additional examination was made after 2 months. Visual acuity (Log MAR) was assessed at every visit. Rate of healing (% of covered area) and subjective sensation of discomfort (scale 0–5) were assessed in the 1st 3 visits. Residual manifest cylinder (D) (as a parameter of corneal irregularity) and corneal clarity (epithelial and stromal haze) were assessed from week 1 to month 2. Results During the first 3 days, both groups showed statistically nonsignificant (P > 0.05) results. From week 1 to month 2, eyes receiving D-panthenol showed better vision and less residual cylinder (P < 0.05) at week 1. For all other parameters, and at different examinations, both groups showed a statistically nonsignificant (P > 0.05) difference. Still, eyes receiving D-panthenol showed better values at the majority of the parameters tested. Conclusion D-Panthenol effect on corneal epithelial regeneration is of minimal clinical relevance. A different dosage and a larger sample of patients might reveal a statistical relevance. This trial is registered with https://doi.org/10.1186/ISRCTN81441126.
Collapse
|
|
26
|
A stochastic model of corneal epithelium maintenance and recovery following perturbation. J Math Biol 2018; 78:1245-1276. [PMID: 30478759 DOI: 10.1007/s00285-018-1308-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2018] [Revised: 11/01/2018] [Indexed: 01/15/2023]
Abstract
Various biological studies suggest that the corneal epithelium is maintained by active stem cells located in the limbus, the so-called limbal epithelial stem cell hypothesis. While numerous mathematical models have been developed to describe corneal epithelium wound healing, only a few have explored the process of corneal epithelium homeostasis. In this paper we present a purposefully simple stochastic mathematical model based on a chemical master equation approach, with the aim of clarifying the main factors involved in the maintenance process. Model analysis provides a set of constraints on the numbers of stem cells, division rates, and the number of division cycles required to maintain a healthy corneal epithelium. In addition, our stochastic analysis reveals noise reduction as the epithelium approaches its homeostatic state, indicating robustness to noise. Finally, recovery is analysed in the context of perturbation scenarios.
Collapse
|
|
27
|
Webster A, Chintala SK, Kim J, Ngan M, Itakura T, Panjwani N, Argüeso P, Barr JT, Jeong S, Fini ME. Dynasore protects the ocular surface against damaging oxidative stress. PLoS One 2018; 13:e0204288. [PMID: 30303976 PMCID: PMC6179211 DOI: 10.1371/journal.pone.0204288] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2018] [Accepted: 09/04/2018] [Indexed: 02/04/2023] Open
Abstract
Water soluble "vital" dyes are commonly used clinically to evaluate health of the ocular surface; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal damage stimulates vital dye uptake by individual living cells. Since cell damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. In support of this idea, we show here that application of oxidative stress to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates both dye uptake and endocytosis, and that dye uptake is blocked by co-treatment with three different endocytosis inhibitors. Stress application to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated, and two of the endocytosis inhibitors did not block dye uptake. The exception was Dynasore and its more potent analogue Dyngo-4a, both small molecules developed to target dynamin family GTPases, but also having off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we went on to demonstrate an alternative mechanism. We show that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into the mechanisms of vital dye uptake and point the direction for future study. Significantly, they also suggest that Dynasore and its analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.
Collapse
Affiliation(s)
- Andrew Webster
- USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, United States of America
| | - Shravan K. Chintala
- USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, United States of America
| | - Jasmine Kim
- Program in Biological Sciences, USC Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA, United States of America
| | - Michelle Ngan
- Program in Preventive Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, United States of America
| | - Tatsuo Itakura
- USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, United States of America
| | - Noorjahan Panjwani
- New England Eye Center/Department of Ophthalmology and Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA, United States of America
| | - Pablo Argüeso
- Schepens Eye Research Institute of Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, MA, United States of America
| | - Joseph T. Barr
- The Ohio State University College of Optometry, Columbus, OH, United States of America
| | - Shinwu Jeong
- USC Institute for Genetic Medicine and USC Roski Eye Institute/Department of Ophthalmology, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, United States of America
| | - M. Elizabeth Fini
- USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, United States of America
| |
Collapse
|
|
28
|
Basche M, Kampik D, Kawasaki S, Branch MJ, Robinson M, Larkin DF, Smith AJ, Ali RR. Sustained and Widespread Gene Delivery to the Corneal Epithelium via In Situ Transduction of Limbal Epithelial Stem Cells, Using Lentiviral and Adeno-Associated Viral Vectors. Hum Gene Ther 2018; 29:1140-1152. [PMID: 30070149 DOI: 10.1089/hum.2018.115] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Corneal epithelial dystrophies are typically characterized by symptoms such as pain, light sensitivity, and corneal opacification leading to impaired vision. The development of gene therapy for such conditions has been hindered by an inability to achieve sustained and extensive gene transfer, as the epithelium is highly replicative and has evolved to exclude foreign material. We undertook a comprehensive study in mice aiming to overcome these impediments. Direct injection of lentiviral vector within the stem cell niche resulted in centripetal streaks of epithelial transgene expression sustained for >1 year, indicating limbal epithelial stem cell transduction in situ. The extent of transgene expression varied markedly but at maximum covered 26% of the corneal surface. After intrastromal injection, adeno-associated viral (AAV) vectors were found to penetrate Bowman's membrane and mediate widespread, but transient (12-16 days), epithelial transgene expression. This was sufficient, when applied within a Cre/lox system, to result in recombined epithelium covering up to approximately 80% of the corneal surface. Lastly, systemic delivery of AAV2/9 in neonatal mice resulted in extensive corneal transduction, despite the relative avascularity of the tissue. These findings provide the foundations of a gene therapy toolkit for the corneal epithelium, which might be applied to correction of inherited epithelial dystrophies.
Collapse
Affiliation(s)
- Mark Basche
- 1 Department of Genetics, UCL Institute of Ophthalmology, London, United Kingdom; London, United Kingdom
| | - Daniel Kampik
- 1 Department of Genetics, UCL Institute of Ophthalmology, London, United Kingdom; London, United Kingdom
| | - Satoshi Kawasaki
- 2 Department of Ophthalmology, Kyoto Prefectural University of Medicine , Kyoto, Japan
| | - Matthew J Branch
- 1 Department of Genetics, UCL Institute of Ophthalmology, London, United Kingdom; London, United Kingdom
| | - Martha Robinson
- 1 Department of Genetics, UCL Institute of Ophthalmology, London, United Kingdom; London, United Kingdom
| | | | - Alexander J Smith
- 1 Department of Genetics, UCL Institute of Ophthalmology, London, United Kingdom; London, United Kingdom
| | - Robin R Ali
- 1 Department of Genetics, UCL Institute of Ophthalmology, London, United Kingdom; London, United Kingdom
- 4 NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, United Kingdom
| |
Collapse
|
|
29
|
Hu W, Zhang Y, Tighe S, Zhu YT, Li GG. A New Isolation Method of Human Lacrimal Canaliculus Epithelial Stem Cells by Maintaining Close Association with Their Niche Cells. Int J Med Sci 2018; 15:1260-1267. [PMID: 30275751 PMCID: PMC6158657 DOI: 10.7150/ijms.27705] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/06/2018] [Accepted: 06/30/2018] [Indexed: 02/06/2023] Open
Abstract
Purpose: To investigate whether lacrimal canaliculus epithelial stem cells (LCESC) could be isolated and expanded in vitro. Methods: The lacrimal canaliculus epithelium of 6 patients with limbal stem cell deficiency (LSCD) caused by alkali burn or Stevens Johnson Syndrome were examined by lacrimal endoscope. Cadaveric eyelids were fixed and prepared for cross section and stained with HE and antibodies against PCK, Vim, p63α, SCF and c-Kit. Canaliculus tissue was separated under an operating microscope using a lacrimal probe as an indicator and digested with collagenase A. The clusters of epithelial cells with closely associated stroma were further digested with Trypsin/EDTA to obtain single cells for culture on Matrigel-coated plastic plates in MESCM media. The expression of SCF, c-Kit and p63α was determined by immunostaining. The colony-forming efficiency on 3T3 feeder layers was also measured by calculating the percentage of the clone number divided by the total number cells seeded. Results: The epithelial layers of five out of six inferior lacrimal canaliculi and all the six superior lacrimal canaliculi were visually normal in appearance. Five to fifteen layers of the epithelium in the human lacrimal canaliculi were present with a small, tightly compacted basal layer of cells expressing PCK, p63α, SCF and c-Kit. LCESC were isolated by collagenase A and obtained clonal growth in MESCM. The colony-forming efficiency of LCESC holoclones on a 3T3 feeder layer was 3.2%, compared to 1.9% for those of limbal stem cells (LSC). Conclusions: Herein, we first report that LCESCs can be isolated and have stem cell characteristics, similar to those of LSCs. Such a discovery raises a promising substrate resource of stem cells for LSC reconstruction in LSCD patients.
Collapse
Affiliation(s)
- Weikun Hu
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PRC. 430030
| | - Yuan Zhang
- Tissue Tech, Inc., Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, FL, USA. 33173
| | - Sean Tighe
- Tissue Tech, Inc., Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, FL, USA. 33173
| | - Ying-Tieng Zhu
- Tissue Tech, Inc., Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, FL, USA. 33173
| | - Gui-Gang Li
- Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PRC. 430030.,Tissue Tech, Inc., Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, FL, USA. 33173
| |
Collapse
|
|
30
|
|
|
31
|
Irani YD, Klebe S, McInnes SJP, Jasieniak M, Voelcker NH, Williams KA. Oral Mucosal Epithelial Cells Grown on Porous Silicon Membrane for Transfer to the Rat Eye. Sci Rep 2017; 7:10042. [PMID: 28855664 PMCID: PMC5577150 DOI: 10.1038/s41598-017-10793-1] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2016] [Accepted: 08/15/2017] [Indexed: 01/09/2023] Open
Abstract
Dysfunction of limbal stem cells or their niche can result in painful, potentially sight-threatening ocular surface disease. We examined the utility of surface-modified porous-silicon (pSi) membranes as a scaffold for the transfer of oral mucosal cells to the eye. Male-origin rat oral mucosal epithelial cells were grown on pSi coated with collagen-IV and vitronectin, and characterised by immunocytochemistry. Scaffolds bearing cells were implanted into normal female rats, close to the limbus, for 8 weeks. Histology, immunohistochemistry and a multiplex nested PCR for sry were performed to detect transplanted cells. Oral mucosal epithelial cells expanded on pSi scaffolds expressed the corneal epithelial cell marker CK3/12. A large percentage of cells were p63+, indicative of proliferative potential, and a small proportion expressed ABCG2+, a putative stem cell marker. Cell-bearing scaffolds transferred to the eyes of live rats, were well tolerated, as assessed by endpoint histology. Immunohistochemistry for pan-cytokeratins demonstrated that transplanted epithelial cells were retained on the pSi membranes at 8 weeks post-implant, but were not detectable on the central cornea using PCR for sry. The pSi scaffolds supported and retained transplanted rat oral mucosal epithelial cells in vitro and in vivo and recapitulate some aspects of an artificial stem cell niche.
Collapse
Affiliation(s)
- Yazad D Irani
- Departments of Ophthalmology, Flinders University, Bedford Park, SA, Australia.
| | - Sonja Klebe
- Anatomical Pathology, Flinders University, Bedford Park, SA, Australia
| | - Steven J P McInnes
- Future Industries Institute, University of South Australia, Mawson Lakes, SA, Australia
| | - Marek Jasieniak
- Future Industries Institute, University of South Australia, Mawson Lakes, SA, Australia
| | - Nicolas H Voelcker
- Future Industries Institute, University of South Australia, Mawson Lakes, SA, Australia
- Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
- Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clayton, VIC, Australia
| | - Keryn A Williams
- Departments of Ophthalmology, Flinders University, Bedford Park, SA, Australia
| |
Collapse
|
|
32
|
|
|
33
|
Sartaj R, Zhang C, Wan P, Pasha Z, Guaiquil V, Liu A, Liu J, Luo Y, Fuchs E, Rosenblatt MI. Characterization of slow cycling corneal limbal epithelial cells identifies putative stem cell markers. Sci Rep 2017. [PMID: 28630424 PMCID: PMC5476663 DOI: 10.1038/s41598-017-04006-y] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
In order to identify reliable markers of corneal epithelial stem cells, we employed an inducible transgenic “pulse-chase” murine model (K5Tta × TRE-H2BGFP) to localize, purify, and characterize slow cycling cells in the cornea. The retention of GFP labeling in slowly dividing cells allowed for localization of these cells to the corneal limbus and their subsequent purification by FACS. Transcriptome analysis from slow cycling cells identified differentially expressed genes when comparing to GFP- faster-dividing cells. RNA-Seq data from corneal epithelium were compared to epidermal hair follicle stem cell RNA-Seq to identify genes representing common putative stem cell markers or determinants, which included Sox9, Fzd7, Actn1, Anxa3 and Krt17. Overlapping retention of GFP and immunohistochemical expression of Krt15, ΔNp63, Sox9, Actn1, Fzd7 and Krt17 were observed in our transgenic model. Our analysis presents an array of novel genes as putative corneal stem cell markers.
Collapse
Affiliation(s)
- R Sartaj
- University of Illinois, Chicago, USA
| | - C Zhang
- Weill Cornell Medical College, New York, USA
| | - P Wan
- Weill Cornell Medical College, New York, USA
| | - Z Pasha
- University of Illinois, Chicago, USA
| | | | - A Liu
- Weill Cornell Medical College, New York, USA
| | - J Liu
- Weill Cornell Medical College, New York, USA
| | - Y Luo
- University of Illinois, Chicago, USA
| | - E Fuchs
- The Rockefeller University, New York, USA
| | | |
Collapse
|
|
34
|
Griffith GL, Kasus-Jacobi A, Pereira HA. Bioactive Antimicrobial Peptides as Therapeutics for Corneal Wounds and Infections. Adv Wound Care (New Rochelle) 2017; 6:175-190. [PMID: 28616359 PMCID: PMC5467138 DOI: 10.1089/wound.2016.0713] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2016] [Accepted: 01/30/2017] [Indexed: 02/06/2023] Open
Abstract
Significance: More than 2 million eye injuries and infections occur each year in the United States that leave civilians and military members with reduced or complete vision loss due to the lack of effective therapeutics. Severe ocular injuries and infections occur in varied settings including the home, workplace, and battlefields. In this review, we discuss the potential of developing antimicrobial peptides (AMPs) as therapeutics for the treatment of corneal wounds and infections for which the current treatment options are inadequate. Recent Advances: Standard-of-care employs the use of fluorescein dye for the diagnosis of ocular defects and is followed by the use of antibiotics and/or steroids to treat the infection and reduce inflammation. Recent advances for treating corneal wounds include the development of amniotic membrane therapies, wound chambers, and drug-loaded hydrogels. In this review, we will discuss an innovative approach using AMPs with the dual effect of promoting corneal wound healing and clearing infections. Critical Issues: An important aspect of treating ocular injuries is that treatments need to be effective and administered expeditiously. This is especially important for injuries that occur during combat and in individuals who demonstrate delayed wound healing. To overcome gaps in current treatment modalities, bioactive peptides based on naturally occurring cationic antimicrobial proteins are being investigated as new therapeutics. Future Directions: The development of new therapeutics that can treat ocular infections and promote corneal wound healing, including the healing of persistent corneal epithelial defects, would be of great clinical benefit.
Collapse
Affiliation(s)
- Gina L. Griffith
- Ocular Trauma and Vision Restoration, United States Army Institute of Surgical Research, Fort Sam Houston, Texas
| | - Anne Kasus-Jacobi
- Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
- Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma
| | - H. Anne Pereira
- Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
- Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma
- Department of Pathology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma
- Department of Cell Biology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma
| |
Collapse
|
|
35
|
Vallabh NA, Romano V, Willoughby CE. Mitochondrial dysfunction and oxidative stress in corneal disease. Mitochondrion 2017; 36:103-113. [PMID: 28549842 DOI: 10.1016/j.mito.2017.05.009] [Citation(s) in RCA: 67] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2016] [Revised: 01/23/2017] [Accepted: 05/18/2017] [Indexed: 12/13/2022]
Abstract
The cornea is the anterior transparent surface and the main refracting structure of the eye. Mitochondrial dysfunction and oxidative stress are implicated in the pathogenesis of inherited (e.g. Kearns Sayre Syndrome) and acquired corneal diseases (e.g. keratoconus and Fuchs endothelial corneal dystrophy). Both antioxidants and reactive oxygen species are found in the healthy cornea. There is increasing evidence of imbalance in the oxidative balance and mitochondrial function in the cornea in disease states. The cornea is vulnerable to mitochondrial dysfunction and oxidative stress due to its highly exposed position to ultraviolet radiation and high oxygen tension. The corneal endothelium is vulnerable to accumulating mitochondrial DNA (mtDNA) damage due to the post- mitotic nature of endothelial cells, yet their mitochondrial genome is continually replicating and mtDNA mutations can develop and accumulate with age. The unique physiology of the cornea predisposes this structure to oxidative damage, and there is interplay between inherited and acquired mitochondrial dysfunction, oxidative damage and a number of corneal diseases. By targeting mitochondrial dysfunction in corneal disease, emerging treatments may prevent or reduce visual loss.
Collapse
Affiliation(s)
- Neeru A Vallabh
- Corneal and External Eye Service, St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom; Institute of Ageing and Chronic Disease, Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom
| | - Vito Romano
- Corneal and External Eye Service, St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
| | - Colin E Willoughby
- Corneal and External Eye Service, St. Paul's Eye Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom; Institute of Ageing and Chronic Disease, Department of Eye and Vision Science, University of Liverpool, Liverpool, United Kingdom.
| |
Collapse
|
|
36
|
Kasetti RB, Gaddipati S, Tian S, Xue L, Kao WWY, Lu Q, Li Q. Study of corneal epithelial progenitor origin and the Yap1 requirement using keratin 12 lineage tracing transgenic mice. Sci Rep 2016; 6:35202. [PMID: 27734924 PMCID: PMC5062132 DOI: 10.1038/srep35202] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2016] [Accepted: 09/27/2016] [Indexed: 12/14/2022] Open
Abstract
Key issues in corneal epithelium biology are the mechanism for corneal epithelium stem cells to maintain the corneal epithelial homeostasis and wound healing responses, and what are the regulatory molecular pathways involved. There are apparent discrepancies about the locations of the progenitor populations responsible for corneal epithelial self-renewal. We have developed a genetic mouse model to trace the corneal epithelial progenitor lineages during adult corneal epithelial homeostasis and wound healing response. Our data revealed that the early corneal epithelial progenitor cells expressing keratin-12 originated from limbus, and gave rise to the transit amplifying cells that migrated centripetally to differentiate into corneal epithelial cells. Our results support a model that both corneal epithelial homeostasis and wound healing are mainly maintained by the activated limbal stem cells originating form limbus, but not from the corneal basal epithelial layer. In the present study, we further demonstrated the nuclear expression of transcriptional coactivator YAP1 in the limbal and corneal basal epithelial cells and its essential role for maintaining the high proliferative potential of those corneal epithelial progenitor cells in vivo.
Collapse
Affiliation(s)
- Ramesh Babu Kasetti
- Departments of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, KY 40202, USA
| | - Subhash Gaddipati
- Departments of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, KY 40202, USA
| | - Shifu Tian
- Departments of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, KY 40202, USA
| | - Lei Xue
- Department of Interventional Radiology, Shanghai 10th People’s Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, Shanghai 200092, China
| | - Winston W.-Y. Kao
- Department of Ophthalmology, University of Cincinnati, OH 45267, USA
| | - Qingxian Lu
- Departments of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, KY 40202, USA
- James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202, USA
| | - Qiutang Li
- Departments of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, Louisville, KY 40202, USA
- James Graham Brown Cancer Center, University of Louisville School of Medicine, Louisville, KY 40202, USA
| |
Collapse
|
|
37
|
Understanding Symptoms and Quality of Life in Patients With Dry Eye Syndrome. Ocul Surf 2016; 14:365-76. [DOI: 10.1016/j.jtos.2016.04.005] [Citation(s) in RCA: 61] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2015] [Revised: 02/23/2016] [Accepted: 04/01/2016] [Indexed: 11/21/2022]
|
|
38
|
Reinstein DZ, Gobbe M, Archer TJ, Carp GI. Mechanism for a Rare, Idiosyncratic Complication Following Hyperopic LASIK: Diurnal Shift in Refractive Error Due to Epithelial Thickness Profile Changes. J Refract Surg 2016; 32:364-71. [DOI: 10.3928/1081597x-20160428-05] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2015] [Accepted: 02/04/2016] [Indexed: 11/20/2022]
|
|
39
|
KACHAM S, BIRRU B, PARCHA SR, BAADHE R. Limbal stem cell deficiency: special focus on tracking limbal stem cells. Turk J Biol 2016. [DOI: 10.3906/biy-1507-144] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022] Open
|
|
40
|
Moving epithelia: Tracking the fate of mammalian limbal epithelial stem cells. Prog Retin Eye Res 2015; 48:203-25. [DOI: 10.1016/j.preteyeres.2015.04.002] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Revised: 04/10/2015] [Accepted: 04/16/2015] [Indexed: 12/13/2022]
|
|
41
|
Nishida T, Inui M, Nomizu M. Peptide therapies for ocular surface disturbances based on fibronectin-integrin interactions. Prog Retin Eye Res 2015; 47:38-63. [PMID: 25645519 DOI: 10.1016/j.preteyeres.2015.01.004] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2014] [Revised: 01/18/2015] [Accepted: 01/21/2015] [Indexed: 11/24/2022]
Abstract
The condition of the corneal epithelium is a critical determinant of corneal transparency and clear vision. The corneal epithelium serves as a barrier to protect the eye from external insults, with its smooth surface being essential for its optical properties. Disorders of the corneal epithelium include superficial punctate keratopathy, corneal erosion, and persistent epithelial defects (PEDs). The prompt resolution of these disorders is important for minimization of further damage to the cornea. Currently available treatment modalities for corneal epithelial disorders are based on protection of the ocular surface in order to allow natural healing to proceed. PEDs remain among the most difficult corneal conditions to treat, however. On the basis of characterization of the pathobiology of PEDs at the cell and molecular biological levels, we have strived to develop new modes of treatment for these defects. These treatments rely on two key concepts: provision of a substrate, such as the adhesive glycoprotein fibronectin, for the attachment and migration of corneal epithelial cells, and activation of these cells by biological agents such as the combination of substance P and insulin-like growth factor-1 (IGF-1). Central to both approaches is the role of the fibronectin-integrin system in corneal epithelial wound healing. Determination of the minimum amino acid sequences required for the promotion of corneal epithelial wound closure by fibronectin (PHSRN) and by substance P (FGLM-amide) plus IGF-1 (SSSR) has led to the development of peptide eyedrops for the treatment of PEDs that are free of adverse effects of the parent molecules.
Collapse
Affiliation(s)
- Teruo Nishida
- Ohshima Hospital of Ophthalmology, 11-8 Kamigofuku, Hakata-ku, Fukuoka 812-0036, Japan; Yamaguchi University, 1-1-1 Minami-kogushi, Ube City, Yamaguchi 755-8505, Japan.
| | - Makoto Inui
- Department of Pharmacology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami-kogushi, Ube City, Yamaguchi 755-8505, Japan
| | - Motoyoshi Nomizu
- Laboratory of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
| |
Collapse
|
|
42
|
|
|
43
|
Bron A, Argüeso P, Irkec M, Bright F. Clinical staining of the ocular surface: Mechanisms and interpretations. Prog Retin Eye Res 2015; 44:36-61. [DOI: 10.1016/j.preteyeres.2014.10.001] [Citation(s) in RCA: 90] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2014] [Revised: 10/08/2014] [Accepted: 10/08/2014] [Indexed: 10/24/2022]
|
|
44
|
Gichuhi S, Ohnuma SI, Sagoo MS, Burton MJ. Pathophysiology of ocular surface squamous neoplasia. Exp Eye Res 2014; 129:172-82. [PMID: 25447808 PMCID: PMC4726664 DOI: 10.1016/j.exer.2014.10.015] [Citation(s) in RCA: 91] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2014] [Accepted: 10/17/2014] [Indexed: 12/22/2022]
Abstract
The incidence of ocular surface squamous neoplasia (OSSN) is strongly associated with solar ultraviolet (UV) radiation, HIV and human papilloma virus (HPV). Africa has the highest incidence rates in the world. Most lesions occur at the limbus within the interpalpebral fissure particularly the nasal sector. The nasal limbus receives the highest intensity of sunlight. Limbal epithelial crypts are concentrated nasally and contain niches of limbal epithelial stem cells in the basal layer. It is possible that these are the progenitor cells in OSSN. OSSN arises in the basal epithelial cells spreading towards the surface which resembles the movement of corneo-limbal stem cell progeny before it later invades through the basement membrane below. UV radiation damages DNA producing pyrimidine dimers in the DNA chain. Specific CC → TT base pair dimer transformations of the p53 tumour-suppressor gene occur in OSSN allowing cells with damaged DNA past the G1-S cell cycle checkpoint. UV radiation also causes local and systemic photoimmunosuppression and reactivates latent viruses such as HPV. The E7 proteins of HPV promote proliferation of infected epithelial cells via the retinoblastoma gene while E6 proteins prevent the p53 tumour suppressor gene from effecting cell-cycle arrest of DNA-damaged and infected cells. Immunosuppression from UV radiation, HIV and vitamin A deficiency impairs tumour immune surveillance allowing survival of aberrant cells. Tumour growth and metastases are enhanced by; telomerase reactivation which increases the number of cell divisions a cell can undergo; vascular endothelial growth factor for angiogenesis and matrix metalloproteinases (MMPs) that destroy the intercellular matrix between cells. Despite these potential triggers, the disease is usually unilateral. It is unclear how HPV reaches the conjunctiva.
Collapse
Affiliation(s)
- Stephen Gichuhi
- London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK; Department of Ophthalmology, University of Nairobi, P.O Box 19676-00202, Nairobi, Kenya.
| | - Shin-ichi Ohnuma
- UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK.
| | - Mandeep S Sagoo
- UCL Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK; Moorfields Eye Hospital, 162 City Road, London EC1V 2PD, UK; St. Bartholomew's Hospital, W Smithfield, London EC1A 7BE, UK.
| | - Matthew J Burton
- London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK; Moorfields Eye Hospital, 162 City Road, London EC1V 2PD, UK.
| |
Collapse
|
|
45
|
Yoon JJ, Ismail S, Sherwin T. Limbal stem cells: Central concepts of corneal epithelial homeostasis. World J Stem Cells 2014; 6:391-403. [PMID: 25258661 PMCID: PMC4172668 DOI: 10.4252/wjsc.v6.i4.391] [Citation(s) in RCA: 81] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2014] [Revised: 08/20/2014] [Accepted: 09/01/2014] [Indexed: 02/06/2023] Open
Abstract
A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface
Collapse
|
|
46
|
Griffith GL, Kasus-Jacobi A, Lerner MR, Pereira HA. Corneal wound healing, a newly identified function of CAP37, is mediated by protein kinase C delta (PKCδ). Invest Ophthalmol Vis Sci 2014; 55:4886-95. [PMID: 25028358 DOI: 10.1167/iovs.14-14461] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
PURPOSE The neutrophil-derived granular protein, CAP37, an innate immune system molecule, has antibiotic and immunomodulatory effects on host cells, including corneal epithelial cells. We previously showed that CAP37 modulates corneal epithelial cell migration, adhesion, and proliferation, and that protein kinase C delta (PKCδ) mediates CAP37-induced chemotaxis of these cells. The objective of this study was to investigate the hypothesis that CAP37 facilitates corneal wound healing through the PKC signaling pathway. METHODS The standard "scratch" assay performed on monolayers of corneal epithelial cells was used to measure the in vitro effect of CAP37 on wound closure. In vivo wound healing in response to CAP37 was measured using a mouse corneal epithelium abrasion model. In vitro and in vivo wound closure were monitored over 48 hours. The PKCδ was visualized during wound closure in cell monolayers and corneal epithelium by immunohistochemistry. The importance of PKCδ in CAP37-induced corneal wound healing was assessed by siRNA. RESULTS We found that CAP37 accelerated wound closure in vitro and in vivo. Maximal closure occurred with concentrations of CAP37 between 250 and 500 ng/mL. Topical applications on mouse cornea led to re-epithelialization of the cornea by 24 hours. Immunohistochemistry of in vitro and in vivo wounds revealed a local increase of PKCδ along the wound edge in CAP37-treated cell monolayers and corneas, compared to untreated controls. CAP37-induced corneal wound healing was significantly reduced in vivo upon treatment with PKCδ siRNA. CONCLUSIONS These findings support the hypothesis that CAP37 facilitates corneal wound healing through the activation of PKCδ.
Collapse
Affiliation(s)
- Gina L Griffith
- Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
| | - Anne Kasus-Jacobi
- Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States Oklahoma Center for Neuroscience, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
| | - Megan R Lerner
- Department of Surgery, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
| | - H Anne Pereira
- Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States
| |
Collapse
|
|
47
|
Comparison of the transmembrane mucins MUC1 and MUC16 in epithelial barrier function. PLoS One 2014; 9:e100393. [PMID: 24968021 PMCID: PMC4072602 DOI: 10.1371/journal.pone.0100393] [Citation(s) in RCA: 100] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2014] [Accepted: 05/25/2014] [Indexed: 01/09/2023] Open
Abstract
Membrane-anchored mucins are present in the apical surface glycocalyx of mucosal epithelial cells, each mucosal epithelium having at least two of the mucins. The mucins have been ascribed barrier functions, but direct comparisons of their functions within the same epithelium have not been done. In an epithelial cell line that expresses the membrane-anchored mucins, MUC1 and MUC16, the mucins were independently and stably knocked down using shRNA. Barrier functions tested included dye penetrance, bacterial adherence and invasion, transepithelial resistance, tight junction formation, and apical surface size. Knockdown of MUC16 decreased all barrier functions tested, causing increased dye penetrance and bacterial invasion, decreased transepithelial resistance, surprisingly, disruption of tight junctions, and greater apical surface cell area. Knockdown of MUC1 did not decrease barrier function, in fact, barrier to dye penetrance and bacterial invasion increased significantly. These data suggest that barrier functions of membrane-anchored mucins vary in the context of other membrane mucins, and MUC16 provides a major barrier when present.
Collapse
|
|
48
|
Haddad A, Faria-e-Sousa SJ. Maintenance of the corneal epithelium is carried out by germinative cells of its basal stratum and not by presumed stem cells of the limbus. ACTA ACUST UNITED AC 2014; 47:470-7. [PMID: 24820068 PMCID: PMC4086173 DOI: 10.1590/1414-431x20143519] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2013] [Accepted: 01/28/2014] [Indexed: 12/18/2022]
Abstract
The purpose of this investigation was to analyze the proliferative behavior of rabbit
corneal epithelium and establish if any particular region was preferentially involved
in epithelial maintenance. [3H]-thymidine was injected intravitreally into
both normal eyes and eyes with partially scraped corneal epithelium. Semithin
sections of the anterior segment were evaluated by quantitative autoradiography.
Segments with active replication (on) and those with no cell division (off) were
intermingled in all regions of the tissue, suggesting that the renewal of the
epithelial surface of the cornea followed an on/off alternating pattern. In the
limbus, heavy labeling of the outermost layers was observed, coupled with a few or no
labeled nuclei in the basal stratum. This suggests that this region is a site of
rapid cell differentiation and does not contain many slow-cycling cells. The
conspicuous and protracted labeling of the basal layer of the corneal epithelium
suggests that its cells undergo repeated cycles of replication before being sent to
the suprabasal strata. This replication model is prone to generate label-retaining
cells. Thus, if these are adult stem cells, one must conclude that they reside in the
corneal basal layer and not the limbal basal layer. One may also infer that the basal
cells of the cornea and not of the limbus are the ones with the main burden of
renewing the corneal epithelium. No particular role in this process could be assigned
to the cells of the basal layer of the limbal epithelium.
Collapse
Affiliation(s)
- A Haddad
- Departamento de Biologia Celular e Molecular, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil
| | - S J Faria-e-Sousa
- Departamento de Oftalmologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil
| |
Collapse
|
|
49
|
Tibrewal S, Sarkar J, Jassim SH, Gandhi S, Sonawane S, Chaudhary S, Byun YS, Ivanir Y, Hallak J, Horner JH, Newcomb M, Jain S. Tear fluid extracellular DNA: diagnostic and therapeutic implications in dry eye disease. Invest Ophthalmol Vis Sci 2013; 54:8051-61. [PMID: 24255046 DOI: 10.1167/iovs.13-12844] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
PURPOSE To determine the abundance of extracellular DNA (eDNA) in tear fluid of patients with dry eye disease (DED) and to report clinical outcomes after DNase I eyedrops use to reduce excessive tear fluid eDNA. METHODS Tear fluid was collected from healthy control subjects and patients with DED. The eDNA abundance was determined with the PicoGreen dye assay. The DED symptoms and clinical signs were recorded and correlated with eDNA abundance. Two patients with DED having excessive eDNA in tear fluid were treated with DNase I eyedrops. RESULTS The PicoGreen dye assay measures tear fluid eDNA abundance after a 2-minute incubation time. With longer incubations, admixed cells also contribute to eDNA measurements. The mean (SE) eDNA abundance in healthy control subjects' tear fluid was 1.4 (0.2) μg/mL. The mean (SE) eDNA abundance in tear fluid of patients with nonautoimmune DED, autoimmune DED, and graft versus host disease was significantly higher: the values were 2.9 (0.6), 5.2 (1.2), and 9.1 (2.3) μg/mL, respectively (P < 0.05). In most of these patients, the PicoGreen dye kinetic assay of tear fluid showed an increase in fluorescence signal due to the presence of viable cells in tear fluid. Tear fluid eDNA had the best correlation with corneal Rose Bengal staining (r = 0.55). Treatment of patients having DED with DNase I eyedrops reduced eDNA abundance, abrogated signal increase, and improved comfort. CONCLUSIONS Excessive eDNA is present in tear fluid of patients with dry eyes. A novel therapeutic approach for managing DED may be to measure eDNA abundance in tear fluid with the PicoGreen dye assay and reduce excessive amounts with DNase I eyedrops.
Collapse
Affiliation(s)
- Sapna Tibrewal
- Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, Illinois
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
50
|
Douvaras P, Mort RL, Edwards D, Ramaesh K, Dhillon B, Morley SD, Hill RE, West JD. Increased corneal epithelial turnover contributes to abnormal homeostasis in the Pax6(+/-) mouse model of aniridia. PLoS One 2013; 8:e71117. [PMID: 23967157 PMCID: PMC3742784 DOI: 10.1371/journal.pone.0071117] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2013] [Accepted: 06/24/2013] [Indexed: 01/09/2023] Open
Abstract
We aimed to test previous predictions that limbal epithelial stem cells (LESCs) are quantitatively deficient or qualitatively defective in Pax6+/− mice and decline with age in wild-type (WT) mice. Consistent with previous studies, corneal epithelial stripe patterns coarsened with age in WT mosaics. Mosaic patterns were also coarser in Pax6+/− mosaics than WT at 15 weeks but not at 3 weeks, which excludes a developmental explanation and strengthens the prediction that Pax6+/− mice have a LESC-deficiency. To investigate how Pax6 genotype and age affected corneal homeostasis, we compared corneal epithelial cell turnover and label-retaining cells (LRCs; putative LESCs) in Pax6+/− and WT mice at 15 and 30 weeks. Limbal BrdU-LRC numbers were not reduced in the older WT mice, so this analysis failed to support the predicted age-related decline in slow-cycling LESC numbers in WT corneas. Similarly, limbal BrdU-LRC numbers were not reduced in Pax6+/− heterozygotes but BrdU-LRCs were also present in Pax6+/− corneas. It seems likely that Pax6+/− LRCs are not exclusively stem cells and some may be terminally differentiated CD31-positive blood vessel cells, which invade the Pax6+/− cornea. It was not, therefore, possible to use this approach to test the prediction that Pax6+/− corneas had fewer LESCs than WT. However, short-term BrdU labelling showed that basal to suprabasal movement (leading to cell loss) occurred more rapidly in Pax6+/− than WT mice. This implies that epithelial cell loss is higher in Pax6+/− mice. If increased corneal epithelial cell loss exceeds the cell production capacity it could cause corneal homeostasis to become unstable, resulting in progressive corneal deterioration. Although it remains unclear whether Pax6+/− mice have LESC-deficiency, we suggest that features of corneal deterioration, that are often taken as evidence of LESC-deficiency, might occur in the absence of stem cell deficiency if corneal homeostasis is destabilised by excessive cell loss.
Collapse
Affiliation(s)
- Panagiotis Douvaras
- Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom
| | - Richard L. Mort
- Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom
| | - Dominic Edwards
- Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom
| | - Kanna Ramaesh
- Tennent Institute of Ophthalmology, Gartnaval General Hospital, Glasgow, United Kingdom
| | - Baljean Dhillon
- School of Clinical Sciences, University of Edinburgh, Edinburgh, United Kingdom
| | - Steven D. Morley
- Division of Health Sciences, University of Edinburgh, Edinburgh, United Kingdom
| | - Robert E. Hill
- Medical Research Council Human Genetics Unit, Medical Research Council Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom
| | - John D. West
- Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom
- * E-mail:
| |
Collapse
|