Exosomes and their miRNA/protein profile in keratoconus-derived corneal stromal cells.

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-ß1, 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.

Gross cystic disease fluid protein-15/prolactin-inducible protein as a biomarker for keratoconus disease.

Keratoconus (KC) is a bilateral degenerative disease of the cornea characterized by corneal bulging, stromal thinning, and scarring. The etiology of the disease is unknown. In this study, we identified a new biomarker for KC that is present in vivo and in vitro. In vivo, tear samples were collected from age-matched controls with no eye disease (n = 36) and KC diagnosed subjects (n = 17). Samples were processed for proteomics using LC-MS/MS. In vitro, cells were isolated from controls (Human Corneal Fibroblasts-HCF) and KC subjects (Human Keratoconus Cells-HKC) and stimulated with a Vitamin C (VitC) derivative for 4 weeks, and with one of the three transforming growth factor-beta (TGF-ß) isoforms. Samples were analyzed using real-time PCR and Western Blots. By using proteomics analysis, the Gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP) was found to be the best independent biomarker able to discriminate between KC and controls. The intensity of GCDFP-15/PIP was significantly higher in healthy subjects compared to KC-diagnosed. Similar findings were seen in vitro, using a 3D culture model. All three TGF-ß isoforms significantly down-regulated the expression of GCDFP-15/PIP. Zinc-alpha-2-glycoprotein (AZGP1), a protein that binds to PIP, was identified by proteomics and cell culture to be highly regulated. In this study by different complementary techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for KC disease. It is likely that exploring the GCDFP-15/PIP-AZGP1 interactions will help better understand the mechanism of KC disease.

Beneficial effect of the antioxidant riboflavin on gene expression of extracellular matrix elements, antioxidants and oxidases in keratoconic stromal cells.

BACKGROUND: Keratoconus manifests as a conical protrusion of the cornea and is characterised by stromal thinning. This causes debilitating visual impairment which may necessitate corneal transplantation. Therapeutic targets related to disease mechanisms are currently lacking, as the pathobiology remains unclear. Many pathological features may be manifestations of defects in wound healing and reactive oxygen species (ROS)-associated functions. In a wide range of tissue and cell types, antioxidant exposure has beneficial effects on both of these pathways. This study investigated the effect of treatment with the antioxidant riboflavin on wound healing and ROS-associated functions in keratoconus. METHODS: Stromal cells were isolated from human central keratoconic (n = 3) and normal (n = 3) corneas. Total RNA was extracted and reverse-transcribed into complementary DNA. The gene expression of 22 genes involved in repair (eight normal and four repair-type extracellular matrix constituents) and ROS-associated processes (eight antioxidants and two ROS-synthesising oxidases) was quantified using quantitative polymerase chain reaction. This was also performed on keratoconic stromal cells treated in vitro with riboflavin (n = 3). RESULTS: In stromal cells from untreated keratoconic corneas (compared with untreated normal corneas), there was an up-regulation of 7/12 extracellular matrix elements. Four of eight antioxidants and two of two oxidases were also increased. In treated keratoconic corneas (compared with untreated keratoconic corneas), six out of eight normal extracellular matrix constituents were up-regulated and two of four repair-type molecules were reduced. An increase was also observed in seven out of eight antioxidants and there was a diminution in two out of two oxidases. CONCLUSION: Riboflavin encourages the synthesis of a normal extracellular matrix and reduces reactive oxygen species levels in keratoconus. This supports the occurrence of wound healing and ROS-associated abnormalities in keratoconus. By targeting the causative disease mechanisms, riboflavin may have therapeutic potential in the clinical management of keratoconus.

Mutation altering the miR-184 seed region causes familial keratoconus with cataract.

MicroRNAs (miRNAs) bind to complementary sequences within the 3' untranslated region (UTR) of mRNAs from hundreds of target genes, leading either to mRNA degradation or suppression of translation. We found that a mutation in the seed region of miR-184 (MIR184) is responsible for familial severe keratoconus combined with early-onset anterior polar cataract by deep sequencing of a linkage region known to contain the mutation. The mutant form fails to compete with miR-205 (MIR205) for overlapping target sites on the 3' UTRs of INPPL1 and ITGB4. Although these target genes and miR-205 are expressed widely, the phenotype is restricted to the cornea and lens because of the very high expression of miR-184 in these tissues. Our finding highlights the tissue specificity of a gene network regulated by a miRNA. Awareness of the important function of miRNAs could aid identification of susceptibility genes and new therapeutic targets for treatment of both rare and common diseases.