Decreased Central and Limbal Epithelium Thickness After Corneal Crosslinking Evaluated by Anterior Segment Optic Coherence Tomography

Aim: To Investigate the central cornea, limbal epithelium thickness and stroma thickness after corneal cross-linking by Anterior Segment Optical Coherence Tomography (AS-OCT) and Scheimpflug topography.Methods: Fifteen keratoconus patients treated with cross-linking(CXL) and fifteen untreated keratoconus patients was included to the study. Corneal central, limbal epithelial, stromal and total thickness with was analyzed by using Anterior Segment Optical Coherence Topography (AS-OCT) and keratometric values ​​were analyzed by Scheimpflug topography.Results: There was a statisticallly significant difference between treated and untreated keratoconus patients according to limbal epithelial thickness (LET). Limbal epithelial thickness was 30.7 ± 5.5µm in the treated keratoconus patients and 45.6 ± 11.5µm in the untreated patients (p = 0.04). Central corneal epithelial thickness (CCET) was 38.27±3.5 in the treated group and 60.8 ± 10.9µm in the untreated group. There was a statistically signifficant difference the between two groups (p=0.01). The posterior astigmatism value was 0.7 ± 0.3D in the treated group and 0.9 ± 0.5D in untreated group by Scheimpflug topography (p=0.03).Conclusion: Our study have shown that the central corneal epithelium and limbal epithelium were significantly thinned as a result of corneal cross-linking. Corneal posterior astigmatism value decreased among the keratometric values. Epithelial thickness and limbal thickness alterations detected with Ant-OCT could be useful for monitorizing the keratoconus patients treated with crosslinking and could show the effectivity of the treatment.

Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling

MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses. RNA-seq combined with quantitative proteomics based on multiplexed isobaric tandem mass tag labeling was performed in LECs transfected with miR-146a mimic vs. mimic control. Western blot and immunostaining were used to confirm the expression of some targeted genes/proteins. A total of 251 differentially expressed mRNAs and 163 proteins were identified. We found that miR-146a regulates the expression of multiple genes in different pathways, such as the Notch system. In LECs and organ-cultured corneas, miR-146a increased Notch-1 expression possibly by downregulating its inhibitor Numb, but decreased Notch-2. Integrated transcriptome and proteome analyses revealed the regulatory role of miR-146a in several other processes, including anchoring junctions, TNF-α, Hedgehog signaling, adherens junctions, TGF-β, mTORC2, and epidermal growth factor receptor (EGFR) signaling, which mediate wound healing, inflammation, and stem cell maintenance and differentiation. Our results provide insights into the regulatory network of miR-146a and its role in fine-tuning of Notch-1 and Notch-2 expressions in limbal epithelium, which could be a balancing factor in stem cell maintenance and differentiation.

Role of Jagged1-mediated Notch Signaling Activation in the Differentiation and Stratification of the Human Limbal Epithelium

The Notch signaling pathway plays a key role in proliferation and differentiation. We investigated the effect of Jagged 1 (Jag1)-mediated Notch signaling activation in the human limbal stem/progenitor cell (LSC) population and the stratification of the limbal epithelium in vitro. After Notch signaling activation, there was a reduction in the amount of the stem/progenitor cell population, epithelial stratification, and expression of proliferation markers. There was also an increase of the corneal epithelial differentiation. In the presence of Jag1, asymmetric divisions were decreased, and the expression pattern of the polarity protein Par3, normally present at the apical-lateral membrane of basal cells, was dispersed in the cells. We propose a mechanism in which Notch activation by Jag1 decreases p63 expression at the basal layer, which in turn reduces stratification by decreasing the number of asymmetric divisions and increases differentiation.

A single cell atlas of human cornea that defines its development, limbal stem and progenitor cells and the interactions with the limbal niche

SummaryTo study the development and composition of human ocular surface, we performed single cell (sc) RNA-Seq at key embryonic, fetal and adult stages and generated the first atlas of the corneal cell types from development to adulthood. Our data indicate that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of proliferative epithelial progenitors, which predate the formation of limbal niche by a few weeks. Bioinformatic comparison of adult cell clusters identified GPHA2, a novel cell-surface marker for quiescent limbal stem cells (qLSCs), whose function is to maintain qLSCs self-renewal. Combining scRNA- and ATAC-Seq analysis, we identified multiple upstream regulators for qLSCs and transit amplifying (TA) cells and demonstrated a close interaction between the immune cells and epithelial stem and progenitor cells in the cornea. RNA-Seq analysis indicated loss of qLSCs and acquisition of proliferative limbal basal epithelial progenitor markers during ex vivo limbal epithelial cell expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium as two key changes underlying the disease phenotype. Our scRNA- and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining pathways/genes that can lead to improvement in ex vivo expansion and differentiation methods for cell based replacement therapies and better understanding and treatment of ocular surface disorders.Key findingsscRNA-Seq of adult human cornea and conjunctiva reveals the signature of various ocular surface cell populationsscRNA-Seq of human developing cornea identifies stage-specific definitions of corneal epithelial, stromal and endothelial layersscRNA-Seq analysis results in identification of novel markers for qLSCs and TA cellsCombined scRNA- and ATAC-Seq analysis reveals key transcriptional networks in qLSCs and TA cells and close interactions with immune cellsExpansion of limbal epithelium results in downregulation of qLSCs and acquisition of proliferative limbal epithelial progenitor markersscRNA-Seq of keratoconus corneas reveals activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epitheliumGraphical abstractSchematic presentation of main techniques and findings presented in this manuscript.

Protection of Corneal Limbus from Riboflavin Prevents Epithelial Stem Cell Loss after Collagen Cross-Linking

Purpose. To investigate whether the protection of corneal limbus from riboflavin exposure during collagen cross-linking (CXL) prevents limbal epithelial stem cell (LESC) loss. Methods. Ten New Zealand white rabbits received an epithelium-off CXL using an accelerated protocol. Seven days before procedure, 5-bromo-2-deoxyuridine (BrdU) was intraperitoneally injected. During procedure, riboflavin was applied to the corneal surface within a 9 mm diameter retention ring in 5 rabbits, thereby preventing the limbus from riboflavin exposure. In other 5 rabbits, riboflavin was instilled every 2 min, allowing the spillover to the limbus. One day after UVA irradiation, corneas were subjected to histological and molecular assays. Results. There were no differences in corneal thickness and epithelial healing between the groups. The numbers of BrdU-labelled and p63+ limbal epithelial cells were markedly reduced in the group without a ring, but significantly increased when a ring was used. Robust expression of CK3/12 was observed in the limbal epithelium in the group with a ring. The mRNA levels of ABCG2, FGF2, IL-1β, and IL-6 were significantly increased in the corneas with a ring. Conclusions. Protection of limbus from riboflavin during CXL was effective in preserving LESCs. However, inflammation was increased in the cornea treated with riboflavin using a ring.

The Role of Limbal Epithelial Stem Cells in Regulating Corneal (Lymph)angiogenic Privilege and the Micromilieu of the Limbal Niche following UV Exposure

The cornea is a clear structure, void of blood, and lymphatic vessels, functioning as our window to the world. Limbal epithelial stem cells, occupying the area between avascular cornea and vascularized conjunctiva, have been implicated in tissue border maintenance, preventing conjunctivalisation and propagation of blood and lymphatic vessels into the cornea. Defects in limbal epithelial stem cells are linked to corneal neovascularisation, including lymphangiogenesis, chronic inflammation, conjunctivalisation, epithelial abnormalities including the presence of goblet cells, breaks in Bowman’s membrane, persistent epithelial defects and ulceration, ocular surface squamous neoplasia, lipid keratopathy, pain, discomfort, and compromised vision. It has been postulated that pterygium is an example of focal limbal deficiency. Previous reports showing changes occurring in limbal epithelium during pterygium pathogenesis suggest that there is a link to stem cell damage. In this light, pterygium can serve as a model disease of UV-induced stem cell damage also characterised by corneal blood and lymphangiogenesis. This review focuses on the role of corneal and limbal epithelial cells and the stem cell niche in maintaining corneal avascularity and corneal immune privilege and how this may be deregulated following UV exposure. We present an overview of the PUBMED literature in the field as well as recent work from our laboratories.