bromochloroacetic-acid and Keratoconus

Keratoconus (KC) is an ocular disorder with a multifactorial origin. Transcriptomic analyses (RNA-seq) revealed deregulations of coding (mRNA) and non-coding RNAs (ncRNAs) in KC, suggesting that mRNA-ncRNA co-regulations can promote the onset of KC. The present study investigates the modulation of RNA editing mediated by the adenosine deaminase acting on dsRNA (ADAR) enzyme in KC.. The level of ADAR-mediated RNA editing in KC and healthy corneas were determined by two indexes in two different sequencing datasets. REDIportal was used to localize known editing sites, whereas new putative sites were de novo identified in the most extended dataset only and their possible impact was evaluated. Western Blot analysis was used to measure the level of ADAR1 in the cornea from independent samples.. KC was characterized by a statistically significant lower RNA-editing level compared to controls, resulting in a lower editing frequency, and less edited bases. The distribution of the editing sites along the human genome showed considerable differences between groups, particularly relevant in the chromosome 12 regions encoding for Keratin type II cluster. A total of 32 recoding sites were characterized, 17 representing novel sites. JUP, KRT17, KRT76, and KRT79 were edited with higher frequencies in KC than in controls, whereas BLCAP, COG3, KRT1, KRT75, and RRNAD1 were less edited. Both gene expression and protein levels of ADAR1 appeared not regulated between diseased and controls.. Our findings demonstrated an altered RNA-editing in KC possibly linked to the peculiar cellular conditions. The functional implications should be further investigated.

Topics: Adenosine Deaminase; Genomics; Humans; Keratins; Keratoconus; RNA; RNA Editing; RNA, Messenger; Transcriptome

Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis (KRT16, KRT14, SPRR1A, SPRR2A, SPRR3, TGM1 and TGM5) and in oxidative stress pathways (NRF2, HMOX1 and HMOX2), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium.

Topics: Cornea; Cornified Envelope Proline-Rich Proteins; Epithelium, Corneal; Humans; Keratins; Keratoconus; NF-E2-Related Factor 2; Oxidative Stress

To evaluate histopathological changes induced in keratoconic corneas after implantation of Intacs intracorneal ring segments (Addition Technology, Inc.).. Departments of Ophthalmology and Pathology, Hospital Pellegrin, Bordeaux, France.. This retrospective study included 8 keratoconic, contact-lens-intolerant eyes of 8 patients who had penetrating keratoplasty (PKP) after removal of Intacs inserts because of a poor refractive outcome or insert extrusion. Light microscopy was performed on all specimens after conventional staining. Immunohistochemistry was performed to identify cell types located next to the tunnel using AE1/AE3 cytokeratins, CD34, vimentin, collagen IV, and alpha-smooth muscle actin monoclonal antibodies.. Conventional histology showed hypoplasia of the epithelium immediately surrounding the channel. There was no evidence of an inflammatory response or foreign-body granuloma. Keratocyte density was decreased above and below the tunnel, and collagen IV synthesis was seen in the scar area. All samples stained negatively with alpha-smooth muscle actin, indicating that myofibroblasts were not present. These changes were no longer visible when PKP was performed more than 6 months after Intacs explantation.. Intacs induced keratocyte apoptosis, probably through a switch to a collagenous synthetic phenotype. Although histological changes seem to be entirely reversible after implant removal, longer follow-up is necessary to determine whether they accelerate corneal thinning and keratoconus progression via apoptosis and release of metalloprotease.

Topics: Actins; Adult; Antigens, CD34; Apoptosis; Collagen Type IV; Corneal Stroma; Device Removal; Disease Progression; Female; Fibroblasts; Humans; Immunohistochemistry; Keratins; Keratoconus; Keratoplasty, Penetrating; Male; Middle Aged; Postoperative Complications; Prostheses and Implants; Prosthesis Implantation; Retrospective Studies; Vimentin

The purpose of this study is to identify corneal proteins differentially expressed between keratoconus and normal epithelial samples. Proteins from the corneal epithelium were isolated from 6 keratoconus and 6 myopia patients (controls) and separated by 2D-gel electrophoresis. Six % and 12% SDS-PAGE gels were used to separate low and high molecular weight proteins. Gels were silver stained and protein spots were defined by Melanie II software. The proteins that were most altered in expression comparing keratoconus and controls were extracted, trypsin-digested, and identified by mass spectroscopy. Approximately 200-500 protein spots were detected on each gel. Nineteen spots were identified as differentially expressed between keratoconus and reference epithelium including cytokeratin 3 (< 7.8 fold), gelsolin (1.6 fold), S100A4 (1.9 fold), and enolase 1 (0.72 fold). Another identified protein found at very high levels was cytokeratin 12. Gelsolin, cytokeratin 3, and cytokeratin 12 have previously been described to be involved in other corneal diseases. Three proteins, gelsolin, alpha enolase, and S100A4 were identified to be differentially expressed in keratoconus compared to reference epithelium and thus may be involved in the pathogenesis.

Topics: Biomarkers; Case-Control Studies; Electrophoresis, Gel, Two-Dimensional; Epithelium, Corneal; Eye Proteins; Gelsolin; Gene Expression Profiling; Humans; Keratins; Keratoconus; Oligonucleotide Array Sequence Analysis; Phosphopyruvate Hydratase; Pilot Projects; Proteomics; S100 Calcium-Binding Protein A4; S100 Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Keratoconus involves thinning and central protuberance of the cornea, scarring and significantly decreased vision. It is one of the major causes of corneal transplantation in this country, but the etiology of this disorder is unclear. In the present study stromal keratocytes were isolated and cultured from normal and keratoconus human corneas. Consistent with the phenotype of cornea thinning, we observed an increased gelatinolytic activity in keratoconus cultures. Characterization of enzyme properties in these cells suggested that gelatinase (type IV collagenase) was responsible for the majority of proteolytic activity found in this system. This elevated gelatinolytic activity was present in spite of lower amounts of total protein being produced by the keratoconus cultures.

Topics: Cells, Cultured; Cornea; Electrophoresis, Gel, Two-Dimensional; Gelatinases; Keratins; Keratoconus; Pepsin A; Protein C; Substrate Specificity