Role of Decorin in Posterior Capsule Opacification and Eye Lens Development (original) (raw)
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Journal of Cellular and Molecular Medicine, 2013
Injury to lens epithelial cells (LECs) leads to epithelial-mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1a/2b suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of a-smooth muscle actin (a-SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1a/2b following EMT/PCO formation. Expression of Tpm1a/2b was up-regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non-cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2b mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1a/2b, as evidenced by increased expression of a-SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm-based inhibitors for postponing PCO and cataractogenesis.
Investigative Opthalmology & Visual Science
PURPOSE. To evaluate the inflammatory cytokine, growth factors, extracellular matrix (ECM) remodeling genes, profibrotic and antifibrotic molecules in patients undergoing glaucoma filtration surgery (GFS). Additionally, the effect of preoperative antiglaucoma medications (AGMs) and postoperative bleb status were related to these parameters. METHODS. Tenon's tissue and aqueous humour (AH) were collected from 207 patients undergoing GFS with primary open-angle glaucoma (POAG) (n ¼ 77), primary angle-closure glaucoma (PACG) (n ¼ 62), and cataract controls (n ¼ 68). Monocyte chemoattractant protein-1 (MCP-1), connective tissue growth factor (CTGF), transforming growth factor b1/2 (TGF-b1/2), lysyl oxidase (LOX), lysyl oxidase L2 (LOXL2), elastin (ELN), collagen type 1 a 1 (COL1A1), secreted protein acidic and rich in cysteine (SPARC), a-smooth muscle actin (a-SMA), and decorin (DCN) were determined in tenon's tissue by real-time PCR and in AH using ELISA. RESULTS. A significant increase was observed in the transcripts of MCP-1, TGF-b2, and SPARC in POAG and PACG (P < 0.05); CTGF, TGF-b1, LOX, LOXL2, ELN, COL1A1, and a-SMA in PACG (P < 0.05) compared with control. DCN transcript was significantly decreased in POAG and PACG (P < 0.05) compared with control. The protein levels of CTGF, TGF-b1/b2, ELN, SPARC, and LOXL2 was significantly elevated in POAG and PACG (P < 0.05); DCN was decreased (P < 0.05) compared with control. These parameters showed significant association with duration of preoperative AGMs and postoperative bleb status. CONCLUSIONS. This study demonstrates increased expression of growth factors and ECM molecules, both at protein and transcript levels in GFS patients. A decreased DCN in AH seems striking, and if restored might have a therapeutic role in minimizing postoperative scarring to improve GFS outcome.
Cells Tissues Organs, 2005
differentiation. While members of the transforming growth factor (TGF ) superfamily have also been implicated to play a role in lens fi ber differentiation, inappropriate TGF  signaling in the anterior lens epithelial cells results in an epithelial-mesenchymal transition (EMT) that bears morphological and molecular resemblance to forms of human cataract, including anterior subcapsular (ASC) and posterior capsule opacifi cation (PCO; also known as secondary cataract or after-cataract), which occurs after cataract surgery. Numerous in vitro and in vivo
International Journal of Molecular Sciences
Cataracts are the leading cause of blindness worldwide. Although surgery is a successful method to restore vision loss due to cataracts, post-surgical complications can occur, such as secondary cataracts, also known as posterior capsular opacification (PCO). PCO arises when lens epithelial cells (LEC) are left behind in the capsular bag following surgery and are induced to undergo epithelial to mesenchymal transition (EMT). Following EMT, LEC morphology and phenotype are altered leading to a loss of transparency and vision. Transforming growth factor (TGF)-β-induced signaling through both canonical, TGF-β/Smad, and non-canonical, β-catenin/Wnt and Rho/ROCK/MRTF-A, pathways have been shown to be involved in lens EMT, and thus PCO. However, the interactions between these signaling pathways in the lens have not been thoroughly explored. In the current study we use rat LEC explants as an ex vivo model, to examine the interplay between three TGF-β-mediated pathways using α-smooth muscle ...
PURPOSE: Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a key pathologic mechanism underlying cataract. Two members of the transforming growth factor-β (TGFβ) superfamily, TGFβ and bone morphogenetic protein-7 (BMP-7) have functionally distinct roles in EMT. While TGFβ is a potent inducer of EMT, BMP-7 counteracts the fibrogenic activity of TGFβ. We examine the modulating effect of BMP-7 on TGFβ-induced EMT in LECs. METHODS: Rat lens epithelial explants were treated exogenously with TGFβ2 alone or in combination with BMP-7 for up to 5 days. Expression levels of E-cadherin, β-catenin, α-smooth muscle actin (α-SMA), and phosphorylated downstream Smads were determined using immunofluorescence and Western blotting. Reverse transcriptase quantitative PCR (RT-qPCR) was used to study gene expression levels of EMT markers and downstream BMP target genes, including the Inhibitors of differentiation (Id). RESULTS: Transforming growth factor-β2 induced LECs to transdifferentiate into myofibroblastic cells. Addition of BMP-7 suppressed TGFβ2-induced α-SMA protein levels and mesenchymal gene expression, with retention of E-cadherin and β-catenin expression to the cell membrane. Addition of BMP-7 prevented lens capsular wrinkling and cellular loss associated with TGFβ2-induced EMT over the 5-day treatment period. The inhibitory effect of BMP-7 was accompanied by an early induction of pSmad1/5 and suppression of TGFβ2-induced pSmad2/3. Treatment with TGFβ2 alone suppressed gene expression of Id2/3 and addition of BMP-7 restored Id2/3 expression. CONCLUSIONS: Exogenous administration of BMP-7 abrogated TGFβ2-induced EMT in rat lens epithelial explants. Understanding the complex interplay between the TGFβ- and BMP-7-associated Smad signaling pathways and their downstream target genes holds therapeutic promise in cataract prevention.
Investigative Opthalmology & Visual Science
PURPOSE. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a key pathologic mechanism underlying cataract. Two members of the transforming growth factor-b (TGFb) superfamily, TGFb and bone morphogenetic protein-7 (BMP-7) have functionally distinct roles in EMT. While TGFb is a potent inducer of EMT, BMP-7 counteracts the fibrogenic activity of TGFb. We examine the modulating effect of BMP-7 on TGFb-induced EMT in LECs. METHODS. Rat lens epithelial explants were treated exogenously with TGFb2 alone or in combination with BMP-7 for up to 5 days. Expression levels of E-cadherin, b-catenin, a-smooth muscle actin (a-SMA), and phosphorylated downstream Smads were determined using immunofluorescence and Western blotting. Reverse transcriptase quantitative PCR (RT-qPCR) was used to study gene expression levels of EMT markers and downstream BMP target genes, including the Inhibitors of differentiation (Id). RESULTS. Transforming growth factor-b2 induced LECs to transdifferentiate into myofibroblastic cells. Addition of BMP-7 suppressed TGFb2-induced a-SMA protein levels and mesenchymal gene expression, with retention of E-cadherin and b-catenin expression to the cell membrane. Addition of BMP-7 prevented lens capsular wrinkling and cellular loss associated with TGFb2-induced EMT over the 5-day treatment period. The inhibitory effect of BMP-7 was accompanied by an early induction of pSmad1/5 and suppression of TGFb2induced pSmad2/3. Treatment with TGFb2 alone suppressed gene expression of Id2/3 and addition of BMP-7 restored Id2/3 expression. CONCLUSIONS. Exogenous administration of BMP-7 abrogated TGFb2-induced EMT in rat lens epithelial explants. Understanding the complex interplay between the TGFb-and BMP-7associated Smad signaling pathways and their downstream target genes holds therapeutic promise in cataract prevention.
TGF-β2–Induced Matrix Modification and Cell Transdifferentiation in the Human Lens Capsular Bag
Investigative Ophthalmology & Visual Science
To study the role of TGF-beta2 in posterior capsule opacification (PCO) and to determine whether CAT-152 (lerdelimumab), a fully human monoclonal antibody that neutralizes the effect of TGF-beta2, can also provide therapeutic benefit for PCO. In vitro capsular bags were prepared from human donor eyes and maintained in a 5% CO(2) atmosphere at 35 degrees C. To investigate expression of active TGF-beta2, capsular bags were incubated in serum-free EMEM for 2, 28, or more than 100 days and analyzed by ELISA (n > or = 4 at each time point). To study underlying mechanisms, match-pair experiments were also performed, so that the medium was supplemented with 0, 1 or 10 ng/mL TGF-beta2 with or without 10 microg/mL CAT-152 (n = 4 in all cases). On-going observations were by phase-contrast microscopy. In addition, donor material from patients who had undergone cataract surgery was analyzed. Cellular architecture was examined by fluorescence cytochemistry. Expression of matrix metalloprotein...
Investigative Opthalmology & Visual Science
PURPOSE. Transforming growth factor-b-induced epithelial-mesenchymal transition (EMT) is one of the main causes of posterior capsular opacification (PCO) or secondary cataract; however, the signaling events involved in TGF-b-induced PCO have not been fully characterized. Here, we focus on examining the role of b-catenin/cyclic AMP response element-binding protein (CREB)-binding protein (CBP) and b-catenin/T-cell factor (TCF)dependent signaling in regulating cytoskeletal dynamics during TGF-b-induced EMT in lens epithelial explants. METHODS. Rat lens epithelial explants were cultured in medium M199 in the absence of serum. Explants were treated with TGF-b2 in the presence or absence of the b-catenin/CBP interaction inhibitor, ICG-001, or the b-catenin/TCF interaction inhibitor, PNU-74654. Western blot and immunofluorescence experiments were carried out and analyzed. RESULTS. An increase in the expression of fascin, an actin-bundling protein, was observed in the lens explants upon stimulation with TGF-b, and colocalized with F-actin filaments. Inhibition of b-catenin/CBP interactions, but not b-catenin/TCF interactions, led to a decrease in TGF-b-induced fascin and stress fiber formation, as well as a decrease in the expression of known markers of EMT, a-smooth muscle actin (a-SMA) and matrix metalloproteinase 9 (MMP9). In addition, inhibition of b-catenin/CBP-dependent signaling also prevented TGF-binduced downregulation of epithelial cadherin (E-cadherin) in lens explants. CONCLUSIONS. We show that b-catenin/CBP-dependent signaling regulates fascin, MMP9, and a-SMA expression during TGF-b-induced EMT. We demonstrate that b-catenin/CBP-dependent signaling is crucial for TGF-b-induced EMT in the lens.
Invest Ophthalmol Visual Sci, 2007
PURPOSE. This report presents a novel model for studies of extracellular matrix (ECM) in posterior capsular opacification (PCO) in vitro. Lens epithelial cells (LEC) were cultured with an intraocular lens (IOL) on a surface of type IV collagen in an evaluation of the importance of the ECM-cell interaction in formation of PCO. Abnormal migration, proliferation, and expression of proteins associated with the epithelial-to-mesenchymal transition (EMT) that characterizes PCO were observed in the presence and absence of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine), which regulates matrix-cell interactions. METHODS. The model for PCO in vitro consisted of an IOL placed on a membrane coated with collagen IV, a major constituent of the lens capsule. LECs from the lenses of wild-type (WT) and SPARC-null (SP-null) mice were cultured in the presence or absence of 10 ng/mL TGF-2 and 20 g/mL recombinant human SPARC (rhSP) for up to 6 days. The migration of LECs was quantified. Labeling with BrdU and the measurement of DNA synthesis were assays for cell proliferation. Expression of the EMT markers, collagen type I, fibronectin, and ␣-smooth muscle actin were assessed using immunocytochemistry or Western immunoblots. RESULTS. LEC migration, proliferation, and the synthesis of EMT markers were enhanced in SP-null compared with WT LECs. TGF-2 inhibited the migration and proliferation of both WT and SP-null LECs in the presence of rhSP. TGF-2 increased the production of collagen type I, fibronectin, and ␣-SMA. The responses of SP-null LECs were rescued by the addition of recombinant human (rh)SP. CONCLUSIONS. A simple IOL culture system was useful for the evaluation of the effects of SPARC and TGF-2 on PCO in vitro. The action of TGF-2 on LEC migration and proliferation is influenced by SPARC, a regulator of matrix-cell interactions.
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.