Antioxidants intake and dry eye syndrome: a crossover, placebo-controlled, randomized trial.

PURPOSE: To assess whether an orally administered antioxidant dietary supplement could improve the objective clinical signs and alleviate the subjective symptoms of dry eye syndrome. METHODS: Twenty-four subjects diagnosed with dry eye syndrome were randomized in a crossover, double-blind, controlled, randomized study to receive a placebo or an antioxidants combination (Oxybiane) for 12 weeks. In all subjects, break-up time (BUT) test, Schirmer test, ocular symptoms (sore eyes, burning, itching, sensation of foreign object in the eye, photophobia, sticky eyes, and redness), visual comfort, and general well-being were evaluated weekly. RESULTS: After 12 weeks of supplementation with Oxybiane, both the BUT scores (27.3%+/-8.4% with Oxybiane versus 3.61%+/-4.3% with the placebo, p=0.017) and the Schirmer scores (26.9%+/-14.2% with Oxybiane versus -4.7%+/-3.4% with the placebo, p=0.037) were significantly increased. A significantly improvement was also observed considering subjective clinical symptoms such as burning (p=0.031), itching (p=0.027), sensation of foreign body in eye (p=0.030), and redness (p=0.043). CONCLUSIONS. Supplementation with oral antioxidants can improve both tear stability and quantity but also subjective clinical signs.

Molecular and metabolic retinoid pathways in the human ocular surface.

PURPOSE: To maintain its integrity, the human ocular surface (cornea and conjunctiva) has an absolute requirement for vitamin A and its active derivatives, the retinoic acids. These retinoids regulate transcriptional levels of target genes through the activation of members of a super-family of ligand-dependant nuclear receptors that feature retinoic acid receptors (RAR) alpha, beta, and gamma as well as retinoid X receptors (RXR) alpha, beta, and gamma. The expression patterns of these receptors have been partial characterized in rabbit, mouse, and human cornea and conjunctiva, but systematic tissue and cellular expression of the three RARs and three RXRs had to be completed at the adult human ocular surface. The first objective of our work was to define their expression patterns in term of genes and proteins for total human conjunctiva, cornea, and the major cell types comprising the adult human ocular surface. The second objective was to demonstrate the presence of different enzymes transforming vitamin A to retinoic acid and the functionality of this metabolic pathway in the corneal epithelium. METHODS: Total mRNA was extracted from human total cornea, conjunctiva, corneal epithelial cells (primary culture and established cell line), corneal keratocytes (primary culture), corneal endothelial cells (established cell line), and conjunctival epithelial cells (established cell line) and was submitted to reverse transcription-polymerase chain reaction (RT-PCR) analysis to determine the expression patterns of the RARs and RXRs using specific primers. Immunological staining (via histochemistry and cellular chemistry) experiments were performed to better localize RAR and RXR proteins in the ocular surface at tissue and cellular levels. We also checked mRNA expression of cellular retinol binding proteins (CRBPs) and cellular retinoic acid binding proteins (CRABPs) with the enzymes involved in retinoic acid generation, i.e., alcohol dehydrogenases (ADHs) and retinal dehydrogenases (RALDHs) or degradation (Cyp26 family members). The enzymatic generation of functional retinoids was confirmed using epithelial corneal cells treated with specific inhibitors of retinol metabolism. RESULTS: RAR alpha, RAR gamma, and RXR alpha are expressed in the cornea, conjunctiva, and all of their constitutive cells, whereas RXR gamma and RXR beta were never detected in the cornea or conjunctiva. RAR beta was absent in primary cultures of corneal keratinocytes. ADH3, ADH4, dehydrogenase/reductase (SDR family) 4 (DHRS4), dehydrogenase/reductase (SDR family) 9 (DHRS9), RALDH1, and RALDH3 are expressed in the ocular surface, as were the retinoid-binding proteins CRBP1, CRABP1, and CRABP2. Retinol dehydrogenase 4 (RODH4) was only detected in the conjunctiva. Corneal epithelial cells convert retinol into retinoic acid using an enzymatic pathway. CONCLUSIONS: For the first time, we have established an exhaustive description of the expressions patterns of RARs, RXRs, ADHs, RALDHs, CRBP, and CRABPs in the human ocular surface. Our results for the human ocular surface demonstrated the presence of all the metabolic and molecular actors of the retinoic acid signaling pathway. We also demonstrated the enzymatic conversion of retinol into active retinoids in the corneal environment.

Kruppel-like factor 6 (KLF6) affects the promoter activity of the alpha1-proteinase inhibitor gene.

PURPOSE: Keratoconus is a progressive disease that thins and scars the cornea. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of inhibitors alpha1-proteinase inhibitor (alpha1-PI) and alpha2-macroglobulin (alpha2-M) are reduced. The present study explored the possible involvement in keratoconus of Krüppel-like factor 6 (KLF6), a transcription factor previously described to be essential for the integrity of the corneal epithelium. The transcript and proteins level of KLF6 and its action in regulating the genes affected in keratoconus were examined in this study. METHODS: Semiquantitative RT-PCR, Western blot analysis, immunofluorescence and in situ hybridization were used to investigate the expression of KLF6 mRNA and protein in normal and keratoconus corneas. Modulation by KLF6 of the promoter activity of alpha1-PI, LAP, cathepsin B, and alpha2-M genes was studied after transient transfection of KLF6 expression plasmid into corneal epithelial cells using promoter-reporter gene assays. Chromatin immunoprecipitation (ChIP) assays were performed to confirm the interactions between KLF6 and promoters of the genes affected in keratoconus. RESULTS: A global increased expression of the transcription factor KLF6 in terms of mRNAs and proteins was observed in total cornea and/or the epithelium in a substantial number of the keratoconus specimens. The promoter activity of the human alpha1-PI gene was suppressed by expression of KLF6 in corneal epithelial cells. The ChIP assay confirmed a physical interaction between KLF6 and the alpha1-PI promoter. CONCLUSIONS: Transcription factor KLF6 downregulates the alpha1-PI gene in corneal epithelial cells and may thereby be involved in keratoconus.

Regulation of corneal keratin-12 gene expression by the human Krüppel-like transcription factor 6.

PURPOSE: The keratin-12 (K12) protein is essential for the integrity of the corneal epithelium. This study was conducted to investigate the possible involvement of Krüppel-like factor 6 (KLF6) in the corneal regulation of K12 gene expression, in view of the presence of one KLF6 potential binding site in the human K12 promoter and the known role of KLF6 in regulating keratin gene expression. METHODS: RT-PCR, Western blot analysis, and immunolocalization experiments were used to investigate the expression of KLF6 mRNA and protein in five human total corneas. The same experimental design was used to explore human corneal epithelial (HCE) cells in 20 patients and a HCE cell line. The ability of the KLF6 protein to modulate K12 promoter activity was studied in the HCE cell line, by transient transfections with a KLF6 expression plasmid and promoter-reporter gene assays. Gel-shift assays were performed to confirm the interactions between the KLF6 protein and specific sequences of the K12 promoter. RESULTS: The presence of KLF6 transcripts and proteins in human total corneal extracts was demonstrated. Immunohistofluorescence experiments showed positive staining specifically present in the corneal epithelial layer. KLF6 transcripts and proteins were also present in corneal epithelial cells in 20 patients and the HCE cell line. Transient transfections of KLF6 showed statistical transactivation of the K12 promoter in HCE cells. The gel-shift assay showed a physical interaction between KLF6 and the K12 promoter. CONCLUSIONS: The expression of KLF6 in HCE cells and its role in the regulation of K12 gene expression were demonstrated.