ascorbic-acid and Keratoconus

Keratoconus (KC) is a corneal thinning disorder that leads to severe vision impairment As opposed to corneal transplantation; corneal collagen crosslinking (CXL) is a relatively non-invasive procedure that leads to an increase in corneal stiffness. In order to evaluate the effect of CXL on human corneal stromal cells in vitro, we developed a 3-D in vitro CXL model, using primary Human corneal fibroblasts (HCFs) from healthy patients and Human Keratoconus fibroblasts (HKCs) from KC patients. Cells were plated on transwell polycarbonate membranes and stimulated by a stable vitamin C. CXL was performed using a mixed riboflavin 0.1% PBS solution followed by UVA irradiation. Our data revealed no significant apoptosis in either HCFs or HKCs following CXL. However, corneal fibrosis markers, Collagen III and α-smooth muscle actin, were significantly downregulated in CXL HKCs. Furthermore, a significant downregulation was seen in SMAD3, SMAD7, and phosphorylated SMADs -2 and -3 expression in CXL HKCs, contrary to a significant upregulation in both SMAD2 and Lysyl oxidase expression, compared to HCFs. Our novel 3-D in vitro model can be utilized to determine the cellular and molecular effects on the human corneal stroma post CXL, and promises to establish optimized treatment modalities in patients with KC.

Topics: Apoptosis; Ascorbic Acid; Cell Culture Techniques; Collagen; Cornea; Corneal Stroma; Cross-Linking Reagents; Fibroblasts; Humans; Keratoconus; Photosensitizing Agents; Polycarboxylate Cement; Riboflavin; Ultraviolet Rays

Keratoconus (KC) affects 1:2000 people and is a disorder where cornea thins and assumes a conical shape. Advanced KC requires surgery to maintain vision. The role of oxidative stress in KC remains unclear. We aimed to identify oxidative stress levels between human corneal keratocytes (HCKs), fibroblasts (HCFs) and keratoconus cells (HKCs). Cells were cultured in 2D and 3D systems. Vitamin C (VitC) and TGF-β3 (T3) were used for 4 weeks to stimulate self-assembled extracellular matrix (ECM). No T3 used as controls. Samples were analyzed using qRT-PCR and metabolomics. qRT-PCR data showed low levels of collagen I and V, as well as keratocan for HKCs, indicating differentiation to a myofibroblast phenotype. Collagen type III, a marker for fibrosis, was up regulated in HKCs. We robustly detected more than 150 metabolites of the targeted 250 by LC-MS/MS per condition and among those metabolites several were related to oxidative stress. Lactate levels, lactate/malate and lactate/pyruvate ratios were elevated in HKCs, while arginine and glutathione/oxidized glutathione ratio were reduced. Similar patterns found in both 2D and 3D. Our data shows that fibroblasts exhibit enhanced oxidative stress compared to keratocytes. Furthermore the HKC cells exhibit the greatest level suggesting they may have a myofibroblast phenotype.

Topics: Arginine; Ascorbic Acid; Cell Differentiation; Cells, Cultured; Collagen Type I; Collagen Type III; Collagen Type IV; Cornea; Corneal Keratocytes; Extracellular Matrix; Fibroblasts; Glutathione; Humans; Keratoconus; Lactic Acid; Malates; Metabolomics; Myofibroblasts; Oxidative Stress; Proteoglycans; Pyruvic Acid; Transforming Growth Factor beta3

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-β) 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-β 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.

Topics: Adipokines; Adult; Ascorbic Acid; Biomarkers; Blotting, Western; Carrier Proteins; Cells, Cultured; Chromatography, Liquid; Cornea; Fibroblasts; Gene Expression; Glycoproteins; Humans; Keratoconus; Membrane Transport Proteins; Proteome; Proteomics; Real-Time Polymerase Chain Reaction; Tandem Mass Spectrometry; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3; Vitamins; Young Adult

Keratoconus (KC) is a non-inflammatory disease of the cornea involving structural changes. Oxidative stress is reported to be parts of its pathology, yet the tear antioxidant status contributed by smaller molecule antioxidants can be indicative of the disease. Therefore this study is an attempt to present the status of small molecule antioxidants in KC condition as well as the influence of contact lens wear (CLW) in KC as evaluated in the tear specimen. Tear fluid was collected using schirmer strips from KC with and without CLW (n = 40) with age matched controls (n = 29). Tear fluid antioxidants cysteine, ascorbic acid, glutathione, uric acid and tyrosine were determined by HPLC electrochemical detection. Tear reactive oxygen species was estimated by fluorescence detection using dichlorodihydrofluorescein diacetate (DCF-DA) method. The corneal epithelial mRNA expression of the enzymes, gamma-glutamine cysteinyl synthase (γ-GCS) and gamma-glutamyl transpeptidase (γ-GT) by semiquantitative RT-PCR. Among the five antioxidant molecules estimated, GSH decreased significantly 50.9 ± 9.4 μM in control and 16 ± 5.7 μM in KC (p = 0.015) with increase in tyrosine 13.9 ± 2.6 μM in control, 30 ± 6.4 μM in KC cases (p = 0.022) and uric acid 162 ± 18 μM in control and 210 ± 32 μM (p < 0.00) in KC compared to the controls. The ROS levels were increased significantly, 55.7 ± 16.7 AU in KC and 23.2 ± 5.8 AU in controls (p = 0.023). No significant change in the tear antioxidants studied was observed in KC cases with and without CL wear. However tyrosine levels were increased significantly in CL wear amongst healthy controls compared to controls (p < 0.000). γ-GCS and γ-GT showed no significant change in KC epithelial cells. Though variations were seen in other antioxidants analysed, they had no statistical significance. Tear specimen in KC can indicate the antioxidant status. KC is associated with increased tear levels of tyrosine, uric acid and decrease in GSH apart from increased ROS. Glutathione decreases with increase in oxidative stress and this emphasises the need for antioxidants to balance the redox status in disease management of KC.

Topics: Adult; Antioxidants; Ascorbic Acid; Chromatography, High Pressure Liquid; Contact Lenses; Cornea; Cysteine; Female; gamma-Glutamyltransferase; Glutamate-Cysteine Ligase; Glutathione; Humans; Keratoconus; Male; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tears; Tyrosine; Uric Acid

Topics: Acute Disease; Adrenal Cortex Hormones; Ascorbic Acid; Burns, Chemical; Chronic Disease; Contact Lenses; Cornea; Corneal Injuries; Edema; Eye Diseases; Glaucoma; Glucose; Glycerol; Humans; Hypertonic Solutions; Idoxuridine; Intraocular Pressure; Keratitis, Dendritic; Keratoconus; Ophthalmic Solutions; Povidone; Silicones; Sodium Chloride; Surface-Active Agents; Tears