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Aniridia-related keratopathy: structural changes, signaling pathways and clinical aspects
Umeå University, Faculty of Medicine, Department of Clinical Sciences, Ophthalmology.ORCID iD: 0000-0002-7240-6515
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 [en]
aniridia, aniridia-related keratopathy, PAX6, keratoplasty, fetal cornea, Notch1, Wnt, sonic hedgehog, mTOR, keratocyte
National Category
Ophthalmology
Research subject
ophthalmology
Identifiers
URN: urn:nbn:se:umu:diva-164258ISBN: 978-91-7855-088-3 (print)OAI: oai:DiVA.org:umu-164258DiVA, id: diva2:1362267
Public defence
2019-11-15, Aula Biologica, Biologihuset, vån 2, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2019-10-25 Created: 2019-10-18 Last updated: 2019-10-22Bibliographically approved
List of papers
1. Aniridia-related keratopathy: structural changes in naïve and transplanted corneal buttons
Open this publication in new window or tab >>Aniridia-related keratopathy: structural changes in naïve and transplanted corneal buttons
Show others...
2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 6, article id e0198822Article in journal (Refereed) Published
Abstract [en]

Background: To study structural changes in naive and surgically treated corneas of aniridia patients with advanced aniridia-related keratopathy (ARK).

Methods and findings: Two naive corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation and were now retransplanted and two adult healthy donor control corneas were processed for immunohistochemistry. Antibodies against extracellular matrix components in the stroma and in the epithelial basement membrane (collagen I and IV, collagen receptor alpha 11 integrin and laminin alpha 3 chain), markers of fibrosis, wound healing and vascularization (fibronectin, tenascin-C, vimentin, alpha-SMA and caveolin-1), cell division (Ki-67) and macrophages (CD68) were used. Naive ARK, KLAL 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 by the presence of caveolin-1 and collagen IV in the pannus of all ARK aniridia corneas. The changes observed in decentered and centered transplants were analogous.

Conclusions: Given the similar pathological features of all cases, conditions inherent to the host seem to play an important role on the pathophysiology of the ARK in the long run.

Place, publisher, year, edition, pages
Public Library of Science, 2018
National Category
Ophthalmology
Identifiers
urn:nbn:se:umu:diva-150386 (URN)10.1371/journal.pone.0198822 (DOI)000434786600055 ()29889891 (PubMedID)2-s2.0-85048312123 (Scopus ID)
Available from: 2018-08-06 Created: 2018-08-06 Last updated: 2019-10-18Bibliographically approved
2. Altered Signaling Pathways in Aniridia-Related Keratopathy
Open this publication in new window or tab >>Altered Signaling Pathways in Aniridia-Related Keratopathy
2018 (English)In: Investigative Ophthalmology and Visual Science, ISSN 0146-0404, E-ISSN 1552-5783, Vol. 59, no 13, p. 5531-5541Article in journal (Refereed) Published
Abstract [en]

PURPOSE. To study the Notch1, Wnt/beta-catenin, sonic hedgehog (SHH), and mammalian target of rapamycin (mTOR) cell signaling pathways in naive and surgically treated corneas of aniridia cases with advanced aniridia-related keratopathy (ARK).

METHODS. Two naive corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation, and two adult healthy control corneas were processed for immunohistochemistry in this descriptive study. Antibodies specific against elements of the Notch1 (Notch1; Dlk1; Numb), Wnt/beta-catenin (Wnt5a; Wnt7a; beta-catenin), SHH (glioma-associated oncogene homolog [Gli1]; Hes1), and mTOR (mTOR1; ribosomal protein S6 [rpS6]) signaling pathways were used as well as antibodies against PAX6 and keratin 13 (Krt13).

RESULTS. All ARK corneas presented signs of conjunctivalization and analogous signaling pathway changes in the subepithelial pannus and epithelium, with decreased detection of the Notch1 signaling pathway and an increased presence of the Notch1 inhibitors Numb and Dlk1. Increased detections 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 with normal controls.

CONCLUSIONS. The similarity in pathway alterations found in all ARK corneas, irrespective of limbal stem cell transplantation, further supports the discussion on the role of host-specific factors and limbal stem cell deficiency in ARK.

Place, publisher, year, edition, pages
The Association for Research in Vision and Ophthalmology, 2018
Keywords
aniridia-related keratopathy, signaling pathways, keratoplasty, keratolimbal allograft, limbal stem cell deficiency, PAX6, sonic hedgehog, mTOR, Wnt, Notch1
National Category
Ophthalmology
Identifiers
urn:nbn:se:umu:diva-155119 (URN)10.1167/iovs.18-25175 (DOI)000452609300004 ()30480741 (PubMedID)
Funder
Swedish Research Council, 2015-02438Västerbotten County Council
Available from: 2019-01-08 Created: 2019-01-08 Last updated: 2019-10-18Bibliographically approved
3. Aniridia-related Keratopathy Relevant Cell Signaling Pathways in Human Fetal Corneas
Open this publication in new window or tab >>Aniridia-related Keratopathy Relevant Cell Signaling Pathways in Human Fetal Corneas
(Vietnamese)Manuscript (preprint) (Other academic)
National Category
Ophthalmology
Identifiers
urn:nbn:se:umu:diva-164256 (URN)
Available from: 2019-10-18 Created: 2019-10-18 Last updated: 2019-10-22
4. Cell signaling pathways in human mutant PAX6 corneal cells: an in vitro model for aniridia-related keratopathy
Open this publication in new window or tab >>Cell signaling pathways in human mutant PAX6 corneal cells: an in vitro model for aniridia-related keratopathy
(English)Manuscript (preprint) (Other academic)
National Category
Ophthalmology
Identifiers
urn:nbn:se:umu:diva-164257 (URN)
Available from: 2019-10-18 Created: 2019-10-18 Last updated: 2019-10-22

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