In vitro model suggests oxidative stress involved in keratoconus disease.
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.
Department of Ophthalmology, University of Oklahoma Health Sciences Center, USA.
D
D
Karamichos
Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology Harvard Medical School, Boston, MA, USA.
A E K
AE
Hutcheon
Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, 80 E Concord Street, Boston, MA 02118, USA.
C B
CB
Rich
Departments of Biochemistry and Ophthalmology, Boston University School of Medicine, 80 E Concord Street, Boston, MA 02118, USA.
V
V
Trinkaus-Randall
Division of Signal Transduction/Mass Spectrometry Core, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA.
J M
JM
Asara
Schepens Eye Research Institute/Massachusetts Eye and Ear and the Department of Ophthalmology Harvard Medical School, Boston, MA, USA.
J D
JD
Zieske
D001120:Arginine / Q000378:metabolism; D001205:Ascorbic Acid / Q000494:pharmacology; D002454:Cell Differentiation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000096:biosynthesis; D024061:Collagen Type III / Q000096:biosynthesis; D024141:Collagen Type IV / Q000096:biosynthesis; D003315:Cornea / Q000166:cytology / Q000473:pathology; D060527:Corneal Keratocytes / Q000166:cytology / Q000473:pathology*; D005109:Extracellular Matrix; D005347:Fibroblasts / Q000166:cytology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007640:Keratoconus / Q000473:pathology*; D019344:Lactic Acid / Q000378:metabolism; D008293:Malates / Q000378:metabolism; D055432:Metabolomics; D058628:Myofibroblasts / Q000166:cytology; D018384:Oxidative Stress; D011509:Proteoglycans / Q000096:biosynthesis; D019289:Pyruvic Acid / Q000378:metabolism; D053782:Transforming Growth Factor beta3 / Q000494:pharmacology
D024042:Collagen Type I; D024061:Collagen Type III; D024141:Collagen Type IV; C490948:KERA protein, human; D008293:Malates; D011509:Proteoglycans; D053782:Transforming Growth Factor beta3; D019344:Lactic Acid; C030298:malic acid; D019289:Pyruvic Acid; D001120:Arginine; D005978:Glutathione; D001205:Ascorbic Acid