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Aniridia-related keratopathy relevant cell signaling pathways in human fetal corneas
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.ORCID iD: 0000-0002-7240-6515
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.ORCID iD: 0000-0002-8039-493x
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.
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2022 (English)In: Histochemistry and Cell Biology, ISSN 0948-6143, E-ISSN 1432-119X, Vol. 158, no 2, p. 169-180Article in journal (Refereed) Published
Abstract [en]

We aimed to study aniridia-related keratopathy (ARK) relevant cell signaling pathways [Notch1, Wnt/β-catenin, Sonic hedgehog (SHH) and mTOR] in normal human fetal corneas compared with normal human adult corneas and ARK corneas. We found that fetal corneas at 20 weeks of gestation (wg) and normal adult corneas showed similar staining patterns for Notch1; however 10–11 wg fetal corneas showed increased presence of Notch1. Numb and Dlk1 had an enhanced presence in the fetal corneas compared with the adult corneas. Fetal corneas showed stronger immunolabeling with antibodies against β-catenin, Wnt5a, Wnt7a, Gli1, Hes1, p-rpS6, and mTOR when compared with the adult corneas. Gene expression of Notch1, Wnt5A, Wnt7A, β-catenin, Hes1, mTOR, and rps6 was higher in the 9–12 wg fetal corneas compared with adult corneas. The cell signaling pathway differences found between human fetal and adult corneas were similar to those previously found in ARK corneas with the exception of Notch1. Analogous profiles of cell signaling pathway activation between human fetal corneas and ARK corneas suggests that there is a less differentiated host milieu in ARK.

Place, publisher, year, edition, pages
Springer, 2022. Vol. 158, no 2, p. 169-180
Keywords [en]
Aniridia, Fetal cornea, Adult cornea, Sonic hedgehog, Notch, mTOR, Wnt
National Category
Ophthalmology
Identifiers
URN: urn:nbn:se:umu:diva-164256DOI: 10.1007/s00418-022-02099-9ISI: 000799082100002PubMedID: 35551459Scopus ID: 2-s2.0-85129792626OAI: oai:DiVA.org:umu-164256DiVA, id: diva2:1362238
Funder
Region VästerbottenUmeå UniversityStiftelsen Kronprinsessan Margaretas arbetsnämnd för synskadadeÅke Wiberg Foundation
Note

Originally included in thesis in manuscript form.

Available from: 2019-10-18 Created: 2019-10-18 Last updated: 2023-09-05Bibliographically approved
In thesis
1. Aniridia-related keratopathy: structural changes, signaling pathways and clinical aspects
Open this publication in new window or tab >>Aniridia-related keratopathy: structural changes, signaling pathways and clinical aspects
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Aniridia is a congenital autosomal dominant, bilateral, panocular condition, caused by haploinsufficiency of the Pax6 transcription factor. Aniridia-related keratopathy (ARK) significantly affects vision and quality of life in these patients. ARK is a chronic progressive keratopathy comprising limbal stem cell deficiency associated with impaired epithelial cell adhesion, corneal conjunctivalization, epithelial erosions and corneal vascular pannus that typically only appear after childhood.

The aims were i) to evaluate the structural changes and ii) cell signaling pathways, including the Notch1, Sonic Hedgehog (SHH), mTOR and Wnt/beta-catenin cell signaling pathways in naïve and surgically treated corneas of aniridia cases with advanced ARK and comparing with normal human adult and fetal corneas and iii) to develop a corneal cell culture model of aniridia.

Naïve ARK corneas removed at the time of the first transplantation and ARK corneal buttons removed after a failed keratolimbal allograft and failed centered and decentered transplantations were included. These were compared with normal human adult and fetal (10-11 and 20 weeks) corneas. Sections were studied with immunofluorescence using antibodies against extracellular matrix components in the stroma and in the epithelial basement membrane (collagen I and IV, collagen receptor a11 integrin and laminin a3 chain), markers of fibrosis, wound healing and vascularization (fibronectin, tenascin-C, vimentin, a-SMA and caveolin-1), cell division (Ki-67) and macrophages (CD68); antibodies against Pax6 and keratin 13; and antibodies against elements of the Notch1 (Notch1; Dlk1; Numb), Wnt/beta-catenin (Wnt5a; Wnt7a; beta- catenin), Sonic Hedgehog (Gli1; Hes1) and mTOR (mTOR1; rpS6) signaling pathways. An in vitro cell culture model of mutant PAX6 corneal cells, obtained with CRISPR was created to study the same signaling pathways with Western blot and RT-qPCR.

All ARK corneas and transplanted corneal buttons presented similar histopathological changes with irregular epithelium and disruption or absence of epithelial basal membrane. There was a loss of the orderly pattern of collagen lamellae and absence of collagen I in all ARK corneas. Vascularization was revealed in the pannus of all ARK corneas and the changes observed in decentered and centered transplants were analogous. Furthermore, ARK corneas presented analogous signaling pathways changes in the subepithelial pannus and epithelium, with decreased detection of Notch1 signaling pathway and increased presence of the Notch1 inhibitors Numb and Dlk1. Increased detection of Wnt/beta-catenin (enhanced presence of Wnt5a, Wnt7a and beta-catenin), SHH (detection of GLI1 and HES1) and mTOR (identification of mTOR and rpS6) signaling pathways were found in the subepithelial pannus and epithelium of all ARK corneas, when compared to normal controls. In the mutant PAX6 corneal cells, the signaling pathway changes encountered were similar to those found in the ARK patients. The cell signaling pathway dissimilarities encountered in ARK corneas were similar to the pattern found in human fetal corneas with the exception of Notch1.

The analogous pathological features of all ARK cases and the similarity in pathway alterations found in all ARK corneas irrespective of being naïve or previously submitted to distinct surgical procedures involving transplantation of limbal stem cells or not, and mutant PAX6 corneal cells, advocates an important role for host specific factors in the pathophysiology of ARK. Moreover, the similar pattern found between fetal human corneas and ARK corneas indicates a less differentiated host milieu in ARK. The present results provide the basis for further studies investigating the possibility of modulating these pathways in order to delay or avoid ARK.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2019. p. 55
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2043
Keywords
aniridia, aniridia-related keratopathy, PAX6, keratoplasty, fetal cornea, Notch1, Wnt, sonic hedgehog, mTOR, keratocyte
National Category
Ophthalmology
Research subject
ophthalmology
Identifiers
urn:nbn:se:umu:diva-164258 (URN)978-91-7855-088-3 (ISBN)
Public defence
2019-11-15, Aula Biologica, Biologihuset, vån 2, Umeå, 09:00 (English)
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Supervisors
Available from: 2019-10-25 Created: 2019-10-18 Last updated: 2019-10-22Bibliographically approved

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Vicente, AndréSloniecka, MartaLiu, Jing-XiaByström, BeritDomellöf, Fatima Pedrosa

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