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

Corneal Epithelium Expresses a Variant of P2X7 Receptor in Health and Disease

Improper wound repair of the corneal epithelium can alter refraction of light resulting in impaired vision. We have shown that ATP is released after injury, activates purinergic receptor signaling pathways and plays a major role in wound closure. In many cells or tissues, ATP activates P2X7 receptors leading to cation fluxes and cytotoxicity. The corneal epithelium is an excellent model to study the expression of both the full-length P2X7 form (defined as the canonical receptor) and its truncated forms. When Ca2+ mobilization is induced by BzATP, a P2X7 agonist, it is attenuated in the presence of extracellular Mg2+ or Zn2+, negligible in the absence of extracellular Ca2+, and inhibited by the competitive P2X7 receptor inhibitor, A438079. BzATP enhanced phosphorylation of ERK. Together these responses indicate the presence of a canonical or full-length P2X7 receptor. In addition BzATP enhanced epithelial cell migration, and transfection with siRNA to the P2X7 receptor reduced cell migration. Furthermore, sustained activation did not induce dye uptake indicating the presence of truncated or variant forms that lack the ability to form large pores. Reverse transcription-polymerase chain reaction and Northern blot analysis revealed a P2X7 splice variant. Western blots identified a full-length and truncated form, and the expression pattern changed as cultures progressed from monolayer to stratified. Cross-linking gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X7 mRNA from diabetic corneal epithelium compared to non-diabetic controls and an increased trend in expression of P2X7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X7, which ultimately allows P2X7 to function as a multifaceted receptor that can mediate cell proliferation and migration or cell death

Effect of Fibrin Glue on the Biomechanical Properties of Human Descemet's Membrane

10.1371/journal.pone.0037456PLoS ONE75

Confocal Imaging of the a 6 and /? 4 Integrin Subunits in the Human Cornea With Aging

Purpose. The purpose of this study was to examine changes in the distribution of integrin subunits, a 6 and a 4 , in the normal human cornea with age. Methods. Thirty normal corneas were examined and divided into three groups; corneas from children younger than 2 years, corneas from adults 29 to 70 years, and corneas from adults older than 70 years. The corneas were frozen and the sections were cut, double-stained with monoclonal antibodies to the integrin subunits, and visualized with Texas Red or fluorescein using confocal laser scanning microscopy. Computer imaging was conducted to determine differences. Results. The a 6 subunit was generally localized along the basal and lateral surfaces of basal epithelial cells and projected into Bowman's membrane. The j8 4 subunit was only present along the basal surface. Overall, the major age-related difference was the loss of continuous a 6 and /3 4 subunits along the basal surface of basal epithelial cells. When reconstructed images from corneas of individuals older than 70 years were optically sectioned en face, the a 6 subunit appeared discontinuous. If the same optical images were viewed from corneas of younger individuals, the staining was continuous. The number and distribution of hemidesmosomes along the basal lamina did not change with age in the corneas examined. Conclusions. Using computer imaging associated with confocal laser scanning microscopy, we have demonstrated that there is an age-related change in the localization of the a 6 and /3 4 subunits. Invest Ophthalmol Vis Sci 1993; 34:3103-3109. L he cornea is comprised of five to seven layers of stratified epithelium in which proliferation is limited to the basal layer. Basal cells form hemidesmosomes with the basal lamina that lies over an acellular collagenous region called Bowman's membrane. Increasing evidence demonstrates that extracellular matrix receptors such as integrins play an important role in the formation of cell substrate and cell-cell junctions. Integrins are transmembrane glycoproteins consisting of an alpha and a beta subunit. The ligand specificity of this receptor depends on its heterodimer composition; however, the same integrin can have different ligands in different cell types