ADAM and ADAMTS gene expression in native and wound healing human lens epithelial cells (original) (raw)
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Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury
The American journal of pathology, 1996
Delayed re-epithelialization of the cornea after injury usually precedes stromal ukceration. Previous findings using a rat thermal injury model suggested that re-epithelialization is impeded by products of resident corneal cells, which destroy adbesive structures at the basement membrane zone. In this study, we provide additional evidencefor this concept. Failure to re-epithelialize was found to correlate with an increase in the amounts of gelatinolytic matrix metalloproteinases present in the rat cornea. One of these gelatinases, gelatinase B, is synthesized by the resident corneal cells, and inhibition of its synthesis correlated with inhibition of basement membrane dissolution. The matrix metalloproteinases collagenase and stromelysin are also synthesized by resident corneal cells in thermaly injured corneas of rabbits, but the timing of bulk enzyme synthesis correlated more closely with deposition of repair tissue in the stroma than with failure to re-epithelialize. Nevertheless, in human corneas with repair defects, gelatinase B and collagenase are synthesized by cells in the basal layer of the epithelium directly adjacent to the basement membrane, suggesting that both couldparticipate in dissolution ofthis structure. Importantly, treatment of thermally injured corneas with a synthetic inhibitor of matrix metalloproteinases significantly improved basement membrane integrity. These data support the concept that over-expression of matrix metalloproteinases by resident corneal cells impedes re-epithelialization after some types ofcorneal injury. (Am J Pathol 1996, 149:1287-1302 Corneal ulceration is a devastating disorder that can cause blindness. Ulcers manifest as a breakdown of the collagenous stromal tissue, a process that was once thought to be a simple physical dissolution described as corneal melting. A major change in our understanding of stromal ulceration occurred when this process was shown to be associated with the secretion of type collagen-degrading enzymes from living cells.' In organ culture, collagenolytic activity was shown to be produced by superficial sections of ulcerating cornea containing the epithelial layer and a small amount of anterior stroma. However, the observation that neutrophil infiltration is a hallmark of stromal ulceration suggested that these cells (with their accumulated stores of hydrolytic enzymes) might provide the more important source of collagenase. Experiments showing that chemical injuries do not ulcerate in animals that have been made neutropenic have provided support for this hypothesis.2
Induction of Matrix Metalloproteinases 2 and 9 following Stress to the Lens
Experimental Eye Research, 2000
Matrix metalloproteinase 2 and 9 (MMP-2 and 9, also known as gelatinase A and B) have been implicated in a number of eye diseases, but their possible involvement in lens pathology is yet to be determined. In the present study, we therefore investigated a possible role of matrix metalloproteinases in cataract and posterior capsule opaci®cation. Whole porcine lenses were removed from the eye and cultured in either Eagles Minimum Essential Medium (EMEM) or EMEM supplemented with 1 mM hydrogen peroxide. The medium was sampled and changed every 2 days. On some occasions a sham cataract operation was performed on cultured lenses. The resulting capsular bag was secured to a Petri dish and cultured in EMEM. Culture media from all preparations were analysed for MMP-2 and 9 activity by gelatin zymography. Media samples from lenses which maintained clarity over the 6 day culture period did not display any detectable gelatinolytic activity. However, media from cataractous lenses demonstrated a gelatinolytic band, which had similar molecular weights to the pro-form of MMP-2. In addition to this band, bands with a similar molecular weight to pro-MMP-9 and its dimeric form were also detected in samples obtained from capsular bag preparations within 24 hr. The data presented indicate that normal lenses have undetectable gelatinase activity. However, there is an associated expression of gelatinases with pathological states of the lens, and therefore gelatinase expression could play an important role in cataractogenesis and posterior capsule opaci®cation.
Experimental Eye Research, 2009
Transforming growth factor beta (TGFβ) has been known to play a role in anterior subcapsular cataract (ASC) formation and posterior capsule opacification (PCO), both of which are fibrotic pathologies of the lens. Several models have been utilized to study ASC formation, including the TGFβ1 transgenic mouse model and the ex-vivo rat lens model. A distinct characteristic of ASC development within these models includes the formation of isolated fibrotic plaques or opacities which form beneath the lens capsule. A hallmark feature of ASC formation is the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) into myofibroblasts. Recently, the matrix metalloproteinases (MMPs) have been implicated in the formation of these cataracts through their involvement in EMT. In the present study, we sought to further investigate the role of MMPs in subcapsular cataract formation in a time course manner, through the examination of gene expression and morphological changes which occur during this process. RT-QPCR and immunohistochemical analysis was carried out on lenses treated with TGFβ for a period of 2, 4 and 6 days. Laser capture microdissection (LCM) was utilized to specifically isolate cells within the plaque region and cells from the adjacent epithelium in lenses treated for a 6 day period. Multilayering of LECs was observed as early as day 2, which preceded the presence of alpha smooth muscle actin (α-SMA) immunoreactivity that was evident following 4 days of treatment with TGFβ. A slight reduction in E-cadherin mRNA was detected at day 2, although this was not significant until the day 4 time point. Importantly, our results also indicate an early induction of MMP-9 mRNA following 2 days of TGFβ treatment, whereas MMP-2 was found to be upregulated at the later 4 day time point. Further experiments using FHL 124 cells show an induction in MMP-2 protein levels following treatment with recombinant MMP-9. Together these findings suggest an upstream role for MMP-9 in ASC formation.
Role of Decorin in Posterior Capsule Opacification and Eye Lens Development
Cells
Decorin (DCN) is involved in a variety of physiological and pathological processes. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) has been proposed as a major cause for the development of posterior capsule opacification (PCO) after cataract surgery. We investigated the plausible target gene(s) that suppress PCO. The expression of Dcn was significantly upregulated in rat PCO tissues compared to that observed in the control using a microarray-based approach. LECs treated with fibroblast growth factor (FGF) 2 displayed an enhanced level of DCN expression, while LECs treated with transforming growth factor (TGF)β-2 showed a decrease in DCN expression. The expression of tropomyosin 1 (Tpm1), a marker of lens EMT increased after the addition of TGFβ-2 in human LEC; however, upregulation of Tpm1 mRNA or protein expression was reduced in human LECs overexpressing human DCN (hDCN). No phenotypic changes were observed in the lenses of 8- and 48-week-old transgenic mi...
31 Towards an Integrative View of Corneal Proteomics in Epithelial Wound Healing
2016
Corneal epithelial wound healing is a continuous and multistep process involves cell migration proliferation and differentiation after an injury. Any defect in these processes will result in loss of corneal transparency and function. Around 135 million people are visually impaired, which illustrate the need for better understanding of corneal healing mechanisms and development of efficient ways to accelerate and improve wound healing. Delayed corneal wound healing contains the risk of bacterial infection causes corneal opacity and neovascularization that could lead to corneal blindness. It is therefore important that epithelium should rapidly regenerate after an injury. Reepithelialization involves cell migration proliferation and differentiation to restore the cornea to its highly organized architecture. The molecular mechanisms underlying these processes are yet to be established. Hence the identification of various proteins that could associate with healing process is of great si...
Current Eye Research, 2016
Purpose-There is limited information on region-specific gene expression in the human corneal stroma. In this study, we aimed to investigate the expression profile of the extracellular matrix and adhesion molecules in the normal corneal stroma using laser capture microdissection (LCM) and molecular techniques. Methods-Frozen sections of human cornea without ocular disease were used to isolate the central and peripheral corneal stromal keratocytes by LCM. RNA was extracted from LCMcaptured tissues and the RT 2 Profiler PCR Arrays were used to examine the expression profile of extracellular matrix and adhesion molecules in the central and peripheral stroma. Real-time quantitative PCR was used to quantify gene expression. Proteomic and western blotting (WB) analyses were performed to confirm gene expression at protein level. Function association network was generated via the web tools String and Cytoscape. Results-The gene expression profiling demonstrated that 35 out of the 84 extracellular matrix and adhesion molecules represented in the array were expressed in stromal keratocytes. Among them, 24 genes were not previously described in the corneal stroma. Two genes were found more abundantly expressed in the central stroma than in the periphery: TGFBI, COL6A2 (p < 0.05). ADAMTS13 was detected only in the central stroma. Proteomics and WB analysis confirmed the expression of 10 genes. Functional analysis revealed that most identified genes were presented in a core cluster that had multiple and strong associations with other genes.
Protective effects of matrix metalloproteinase-12 following corneal injury
Journal of Cell Science, 2013
Corneal scarring due to injury is a leading cause of blindness worldwide and results from dysregulated inflammation and angiogenesis during wound healing. Here we demonstrate that the extracellular matrix metalloproteinase MMP12 (macrophage metalloelastase) is an important regulator of these repair processes. Chemical injury resulted in higher expression of the fibrotic markers α-smooth muscle actin and type I collagen, and increased levels of angiogenesis in corneas of MMP12−/− mice compared with corneas of wild-type mice. In vivo, we observed altered immune cell dynamics in MMP12−/− corneas by confocal imaging. We determined that the altered dynamics owed to an altered inflammatory response, with delayed neutrophil infiltration during the first day and excessive macrophage infiltration six days later, mediated by altered expression levels of chemokines CCL2 and CXCL1, respectively. Corneal repair returned to normal upon inhibition of these chemokines. Taken together, these data sh...