Dataset Information

Characterization of Corneal Epithelial Cells in Keratoconus.

ABSTRACT:

Purpose

We studied the cellular characteristics of epithelial cells in the cone and extraconal periphery of corneas in keratoconus eyes.

Methods

This prospective observational study was conducted at Narayana Nethralaya Eye Institute. A total of 83 and 42 eyes with keratoconus and normal topography, respectively, were included in the study. Corneal epithelial cells were collected and analyzed for apoptosis, proliferation, epithelial-mesenchymal transition, and differentiation status using molecular and biochemical tools. Statistical analysis was performed using the Student's t-test.

Results

Corneal epithelial cells from the cone showed significantly higher expression of proapoptotic marker BAX (P < 0.005) compared to controls. Significantly elevated expression of cell cycle markers CYCLIN D1 (P < 0.005) and Ki67 (P < 0.005) were noted in the extraconal region compared to controls. Cells of the cone showed significantly higher ZO-1 (P < 0.005) and lower vimentin (P < 0.005) compared to controls. Significantly lower expression of the differentiation marker CK3/12 (P < 0.05) was observed in cones compared to controls.

Conclusions

Cones of keratoconic corneas show enhanced cell death, poor differentiation, proliferation and epithelial-mesenchymal transition. The cellular changes of the corneal epithelial cells in the cone and extraconal region differ significantly in a keratoconus corneas.

Translational relevance

Characterization of patient-specific corneal epithelial cellular status in keratoconus has the potential to determine the optimal treatment and therapeutic outcomes paving the way towards personalized treatment in the future.

SUBMITTER: Shetty R

PROVIDER: S-EPMC6322712 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Publications

Characterization of Corneal Epithelial Cells in Keratoconus.

Shetty Rohit R Vunnava Krishna Poojita KP Dhamodaran Kamesh K Matalia Himanshu H Murali Subramani S Jayadev Chaitra C Murugeswari Ponnulagu P Ghosh Arkasubhra A Das Debashish D

Translational vision science & technology 20180103 1

<h4>Purpose</h4>We studied the cellular characteristics of epithelial cells in the cone and extraconal periphery of corneas in keratoconus eyes.<h4>Methods</h4>This prospective observational study was conducted at Narayana Nethralaya Eye Institute. A total of 83 and 42 eyes with keratoconus and normal topography, respectively, were included in the study. Corneal epithelial cells were collected and analyzed for apoptosis, proliferation, epithelial-mesenchymal transition, and differentiation statu ...[more]

PMID: 30627477

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Project description:Keratoconus (KC) is a corneal thinning disorder and a leading cause of corneal transplantation worldwide. Exosomes are small, secreted extracellular vesicles (30-150 nm) that mediate cellular communication via their protein, lipid, and nucleic acid content. We aimed to characterize the exosomes secreted by primary corneal fibroblasts from subjects with or without KC. Using human keratoconus stromal fibroblast cells (HKC, n = 4) and healthy stromal fibroblasts (HCF, n = 4), we collected and isolated exosomes using serial ultracentrifugation. Using nanoparticle tracking analysis (NTA) with ZetaView®, we compared the size and concentration of isolated exosomes. Different exosomal markers were identified and quantified using a transmission electron microscope (TEM) (CD81) and Western blot (CD9 and CD63). Exosomal miRNA profiles were determined by qRT-PCR using Exiqon Human panel I miRNA assays of 368 pre-selected miRNAs. Proteomic profiles were determined using a label-free spectral counting method with mass spectrometry. Differential expression analysis for miRNAs and proteins was done using student's t-test with a significance cutoff of p-value ≤0.05. We successfully characterized exosomes isolated from HCFs using several complementary techniques. We found no significant differences in the size, quantity, or morphology between exosomes secreted by HCFs with or without KC. Expression of CD81 was confirmed by immuno-EM, and expression of CD63 and CD9 with western blots in all exosome samples. We detected the expression of 72-144 miRNAs (threshold cycle Ct < 36) in all exosome samples. In HKC-derived exosome samples, miR-328-3p, miR-532-5p, miR-345-5p, and miR-424-5p showed unique expression, while let-7c-5p and miR-665 have increased expression. Protein profiling identified 157 proteins in at least half of the exosome samples, with 38 known exosomal proteins. We identified 12 up- and 2 down-regulated proteins in HKC-derived exosomes. The proteins are involved in membrane-bounded vesicles, cytoskeletal, calcium binding, and nucleotide binding. These proteins are predicted to be regulated by NRF2, miR-205, and TGF-β1, which are involved in KC pathogenesis. We successfully characterized the HKC-derived exosomes and profiled their miRNA and protein contents, suggesting their potential role in KC development. Further studies are necessary to determine if and how these exosomes with differential protein/miRNA profiles contribute to the pathogenesis of KC.

Dataset Information

Characterization of Corneal Epithelial Cells in Keratoconus.

Purpose

Methods

Results

Conclusions

Translational relevance

Publications

Characterization of Corneal Epithelial Cells in Keratoconus.

Similar Datasets

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