Keratoconus (KC) is a corneal thinning disorder that leads to loss of visual acuity through ectasia, opacity, and irregular astigmatism. It is one of the leading indicators for corneal transplantation in the Western countries. KC usually starts at puberty and progresses until the third or fourth decade; however its progression differs among patients. In the keratoconic cornea, all layers except the endothelium have been shown to have histopathological structural changes. Despite numerous studies in the last several decades, the mechanisms of KC development and progression remain unclear. Both genetic and environmental factors may contribute to the pathogenesis of KC. Many previous articles have reviewed the genetic aspects of KC, but in this review we summarize the histopathological features of different layers of cornea and discuss the differentially expressed proteins in the KC-affected cornea. This summary will help emphasize the major molecular defects in KC and identify additional research areas related to KC, potentially opening up possibilities for novel methods of KC prevention and therapeutic intervention.

PurposeKeratoconus (KC) is the most common corneal ectasia. We aimed to determine the differential expression of coding and long noncoding RNAs (lncRNAs) in human corneas affected with KC.MethodsFrom the corneas of 10 KC patients and 8 non-KC healthy controls, 200 ng total RNA was used to prepare sequencing libraries with the SMARTer Stranded RNA-Seq kit after ribosomal RNA depletion, followed by paired-end 50-bp sequencing with Illumina Sequencer. Differential analysis was done using TopHat/Cufflinks with a gene file from Ensembl and a lncRNA file from NONCODE. Pathway analysis was performed using WebGestalt. Using the expression level of differentially expressed coding and noncoding RNAs in each sample, we correlated their expression levels in KC and controls separately and identified significantly different correlations in KC against controls followed by visualization using Cytoscape.ResultsUsing |fold change| ≥ 2 and a false discovery rate ≤ 0.05, we identified 436 coding RNAs and 584 lncRNAs with differential expression in the KC-affected corneas. Pathway analysis indicated the enrichment of genes involved in extracellular matrix, protein binding, glycosaminoglycan binding, and cell migration. Our correlation analysis identified 296 pairs of significant KC-specific correlations containing 117 coding genes enriched in functions related to cell migration/motility, extracellular space, cytokine response, and cell adhesion. Our study highlighted the potential roles of several genes (CTGF, SFRP1, AQP5, lnc-WNT4-2:1, and lnc-ALDH3A2-2:1) and pathways (TGF-β, WNT signaling, and PI3K/AKT pathways) in KC pathogenesis.ConclusionsOur RNA-Seq–based differential expression and correlation analyses have identified many potential KC contributing coding and noncoding RNAs.

The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly impacts the tear film and its components. Proteomic analysis of tear fluid is challenging mainly due to the wide dynamic range of proteins and the small sample volumes. However, recent advancements in mass spectrometry have revolutionized the field of proteomics enabling unprecedented depth, speed, and accuracy, even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass spectrometry workflows for the proteomic analysis of tear fluid collected via Schirmer strips. We were able to establish a method of in-strip protein digestion that identified >3000 proteins in human tear samples from 11 healthy subjects. Our method offers a significant improvement in the number of proteins identified compared to previously reported methods without pooling samples.

In contact with the external environment, the cornea can easily be injured. Although corneal wounds generally heal rapidly, the pain and increased risk of infection associated with a damaged cornea, as well as the impaired healing observed in some individuals, emphasize the need for novel treatments to accelerate corneal healing. We previously demonstrated in epidermal keratinocytes that the glycerol channel aquaporin-3 (AQP3) interacts with phospholipase D2 (PLD2) to produce the signaling phospholipid phosphatidylglycerol (PG), which has been shown to accelerate skin wound healing in vivo. We hypothesized that the same signaling pathway might be operational in corneal epithelial cells. METHODS. We used co-immunoprecipitation, immunohistochemistry, scratch wound healing assays in vitro, and corneal epithelial wound healing assays in vivo to determine the role of the AQP3/PLD2/PG signaling pathway in corneal epithelium. RESULTS. AQP3 was present in human corneas in situ, and AQP3 and PLD2 were coimmunoprecipitated from corneal epithelial cell lysates. The two proteins could also be co-immunoprecipitated from insect cells simultaneously infected with AQP3-and PLD2-expressing baculoviruses, suggesting a likely direct interaction. A particular PG, dioleoylphosphatidylglycerol (DOPG), enhanced scratch wound healing of a corneal epithelial monolayer in vitro. DOPG also accelerated corneal epithelial wound healing in vivo, both in wild-type mice and in a mouse model exhibiting impaired corneal wound healing (AQP3 knockout mice). CONCLUSIONS. These results indicate the importance of the AQP3/PLD2/PG signaling pathway in corneal epithelial cells and suggest the possibility of developing DOPG as a pharmacologic therapy to enhance corneal wound healing in patients.