Molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas.
ABSTRACT The purpose of the study was to determine molecular changes in selected epithelial proteins in human keratoconus (KC) corneas compared to normal corneas.
Two-dimensional (2-D) gel electrophoretic profiles of epithelial cell proteins from normal and keratoconus corneas were compared, and the selected protein spots that showed either up- or downregulation were identified. The desired spots were identified after trypsin digestion and mass spectrometric analysis. Based on the results, two proteins, alpha-enolase and beta-actin, were further analyzed by immunohistochemical and western blot methods, using respective antibodies. To determine the presence of mRNA of the two proteins in the epithelial cells, RT-PCR studies were performed.
On comparison of the 2-D gel electrophoretic protein profiles, two protein spots were identified in normal corneas that were either absent or present at lower levels in keratoconus corneas. The two spots were determined to be alpha-enolase (48 kDa) and beta-actin (42 kDa) by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), and ES-MS/MS mass spectrometric methods. Immunohistochemical analysis revealed that alpha-enolase and beta-actin were present at extremely low levels in the epithelial superficial and wing cells of the keratoconus corneas compared to these cells of normal corneas. 2-D gel electrophoresis followed by western blot analysis revealed relatively greater degradation of the two proteins in the keratoconus corneas compared to normal corneas. RT-PCR analysis showed the mRNA expression of the two proteins in the epithelial cells of both normal and keratoconus corneas.
The results showed relatively low or negligible levels of alpha-enolase and beta-actin in the wing and superficial epithelial cells of keratoconus corneas compared to normal corneas. This was attributed to relatively greater degradation of the two proteins in keratoconus corneas compared to normal corneas.
SourceAvailable from: Murali Subramani[Show abstract] [Hide abstract]
ABSTRACT: The present study was designed to understand the role of inflammatory cytokines secreted by corneal epithelial cells in keratoconus (KC) and the response to treatment with cyclosporine A (CyA). The study involved 129 Indian KC patients clinically graded according to Amsler-Krumeich classification and 20 healthy, nonectatic subjects as controls. Tear levels of matrix metalloproteinase-9 (MMP9), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) were measured using ELISA kits. Gene expression was measured by qPCR in corneal epithelial cells obtained by debridement from subjects undergoing ocular surface surgeries. In addition, epithelial cells were stimulated with TNFα and treated with CyA to study its role on MMP9 expression. Finally, 20 KC patients (27 eyes) with inflammatory symptoms were treated with topical CyA application. We observed that MMP9, TNFα, and IL6 levels were strongly upregulated at the mRNA level in KC patient epithelia. Similarly, tears collected from KC patients exhibited high levels of MMP9 and IL6 protein. Cyclosporine A treatment significantly reduced the mRNA expression levels of IL6 and TNFα in both short- and long-term treatments; however, it reduced MMP9 levels only in long-term treatment in cultured corneal epithelial cells. Subsequent treatment of KC patients with CyA for approximately 6 months reduced tear MMP9 levels and led to local reduction in corneal curvatures as determined by corneal topography maps. The data indicate that corneal epithelium contributes to elevated MMP9 and inflammatory cytokine expression in tears of KC patients. Cyclosporine A treatment reduced MMP9 and inflammatory cytokine levels in an in vitro inflammation model system. In KC patients, CyA treatment reduced MMP9 levels measured in tears with concomitant arrest of disease progression. Therefore, CyA might be a novel treatment strategy in KC patients but requires additional evaluation in larger cohorts. (ClinicalTrials.gov number, NCT01746823.). Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.Investigative Ophthalmology & Visual Science 02/2015; 56(2). DOI:10.1167/iovs.14-14831 · 3.66 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Keratoconus manifests as a conical protrusion of the cornea and is characterised by stromal thinning. This causes debilitating visual impairment which may necessitate corneal transplantation. Therapeutic targets related to disease mechanisms are currently lacking, as the pathobiology remains unclear. Many pathological features may be manifestations of defects in wound healing and reactive oxygen species (ROS)-associated functions. In a wide range of tissue and cell types, antioxidant exposure has beneficial effects on both of these pathways. This study investigated the effect of treatment with the antioxidant riboflavin on wound healing and ROS-associated functions in keratoconus. Stromal cells were isolated from human central keratoconic (n = 3) and normal (n = 3) corneas. Total RNA was extracted and reverse-transcribed into complementary DNA. The gene expression of 22 genes involved in repair (eight normal and four repair-type extracellular matrix constituents) and ROS-associated processes (eight antioxidants and two ROS-synthesising oxidases) was quantified using quantitative polymerase chain reaction. This was also performed on keratoconic stromal cells treated in vitro with riboflavin (n = 3). In stromal cells from untreated keratoconic corneas (compared with untreated normal corneas), there was an up-regulation of 7/12 extracellular matrix elements. Four of eight antioxidants and two of two oxidases were also increased. In treated keratoconic corneas (compared with untreated keratoconic corneas), six out of eight normal extracellular matrix constituents were up-regulated and two of four repair-type molecules were reduced. An increase was also observed in seven out of eight antioxidants and there was a diminution in two out of two oxidases. Riboflavin encourages the synthesis of a normal extracellular matrix and reduces reactive oxygen species levels in keratoconus. This supports the occurrence of wound healing and ROS-associated abnormalities in keratoconus. By targeting the causative disease mechanisms, riboflavin may have therapeutic potential in the clinical management of keratoconus.Clinical and Experimental Optometry 02/2014; 97(4). DOI:10.1111/cxo.12138 · 1.26 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Keratoconus (KC) is characterized by progressive vision loss due to corneal thinning and structural abnormalities. It is hypothesized that KC is caused by deregulated collagen levels and collagen fibril-maturating enzyme lysyl oxidase (LOX). Further, it is currently not understood whether the gene expression deregulated by the corneal epithelium influences KC pathogenesis. We studied (i) the expressions of the LOX, collagen I (COL IA1), collagen IV (COL IVA1), MMP9, and IL6 genes in KC corneal epithelia, (ii) validated their expression levels in patient tissues, and (iii) correlated expression levels with KC disease severity. The primary goal of this study was to evaluate the importance of these genes in the progression of KC. We analyzed the gene expression levels of the key proteins LOX, collagens (COL IA1 and COL IVA1), MMP9, and IL6 in debrided corneal epithelia from a large cohort of KC patients (90 eyes) and compared them to control patients (52 eyes) without KC. We measured the total LOX activity in the tears of KC patients compared to controls. We also correlated the protein expression levels of LOX and collagens by immunohistochemistry (IHC) in primary tissues from KC patients (27 eyes) undergoing keratoplasty compared to healthy donor corneas (15 eyes). We observed a significant reduction in LOX transcript levels in KC corneal epithelia, and LOX activity in KC tears correlated with disease severity. Collagen transcripts were also reduced in KC while MMP9 transcript levels were upregulated and correlated with disease severity. IL6 was moderately increased in KC patients. IHC demonstrated a reduction in the protein expression levels of LOX in the epithelium and collagen IV in the basement membrane of KC patients compared to healthy donor corneas. The data demonstrates that the structural deformity of the KC cornea may be dependent on reduced expressions of collagens and LOX, as well as on MMP9 elevated by the corneal epithelium.Molecular vision 01/2015; · 2.25 Impact Factor