RESUMO
PURPOSE: The epithelial-mesenchymal transition of human lens epithelial cells plays a role in posterior capsule opacification, a fibrotic process that leads to a common type of cataract. Hyaluronic acid has been implicated in this fibrosis. Studies have investigated the role of transforming growth factor (TGF)-ß2 in epithelial-mesenchymal transition. However, the role of TGF-ß2 in hyaluronic acid-mediated fibrosis of lens epithelial cell remains unknown. We here examined the role of TGF-ß2 in the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells. METHODS: Cultured human lens epithelial cells (HLEB3) were infected with CD44-siRNA by using the Lipofectamine 3000 transfection reagent. The CCK-8 kit was used to measure cell viability, and the scratch assay was used to determine cell migration. Cell oxidative stress was analyzed in a dichloro-dihydro-fluorescein diacetate assay and by using a flow cytometer. The TGF-ß2 level in HLEB3 cells was examined through immunohistochemical staining. The TGF-ß2 protein level was determined through western blotting. mRNA expression levels were determined through quantitative real-time polymerase chain reaction. RESULTS: Treatment with hyaluronic acid (1.0 µM, 24 h) increased the epithelial-mesenchymal transition of HLEB3 cells. The increase in TGF-ß2 levels corresponded to an increase in CD44 levels in the culture medium. However, blocking the CD44 function significantly reduced the TGF-ß2-mediated epithelial-mesenchymal transition response of HLEB3 cells. CONCLUSIONS: Our study showed that both CD44 and TGF-ß2 are critical contributors to the hyaluronic acid-mediated epithelial-mesenchymal transition of lens epithelial cells, and that TGF-ß2 in epithelial-mesenchymal transition is regulated by CD44. These results suggest that CD44 could be used as a target for preventing hyaluronic acid-induced posterior capsule opacification. Our findings suggest that CD44/TGF-ß2 is crucial for the hyaluronic acid-induced epithelial-mesenchymal transition of lens epithelial cells.
Assuntos
Movimento Celular , Células Epiteliais , Transição Epitelial-Mesenquimal , Receptores de Hialuronatos , Ácido Hialurônico , Cristalino , Fator de Crescimento Transformador beta2 , Humanos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Ácido Hialurônico/farmacologia , Receptores de Hialuronatos/metabolismo , Fator de Crescimento Transformador beta2/farmacologia , Fator de Crescimento Transformador beta2/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Cristalino/citologia , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Western Blotting , Opacificação da Cápsula/metabolismo , Opacificação da Cápsula/patologia , Reação em Cadeia da Polimerase em Tempo Real , Citometria de Fluxo , Imuno-Histoquímica , Células CultivadasRESUMO
PURPOSE: To investigate the extracellular matrix and cellular components in lens capsules extracted from patients with dead bag syndrome (DBS) through immunohistochemistry. SETTING: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan, and Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah, Salt Lake City, Utah. DESIGN: Immunohistochemical experimental study. METHODS: 9 capsular bag specimens from DBS cases, as well as 2 control specimens from late-postoperative in-the-bag intraocular lens dislocation cases related to previous vitrectomy, pseudoexfoliation, and blunt trauma were included. They were processed for histopathology; unstained sections were obtained from each one and analyzed by immunohistochemistry targeting collagen type IV, laminin, vimentin, collagen type I, and fibronectin. RESULTS: Immunohistochemistry in DBS showed lens capsule stained for basement membrane components. The outer part of the anterior capsule that was split from the inner part was more markedly stained for type IV collagen as compared with the posterior part. Faint staining for fibrous posterior capsular opacification (PCO) components, for example, collagen type I and fibronectin, was detected in limited areas, but the major portion of the capsule was free from these components. Small spotty vimentin-positive materials, suggesting the presence of cell debris, were also detected in limited samples. CONCLUSIONS: Small amounts of fibrotic PCO components were detected in capsules extracted from patients with DBS, but their major parts were free from PCO components. Current findings suggest small amounts of lens epithelial cells were present after surgery and secreted fibrous components before undergoing cell death process.
Assuntos
Colágeno Tipo IV , Colágeno Tipo I , Fibronectinas , Cápsula do Cristalino , Vimentina , Humanos , Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Masculino , Vimentina/metabolismo , Idoso , Feminino , Cápsula do Cristalino/patologia , Cápsula do Cristalino/metabolismo , Pessoa de Meia-Idade , Colágeno Tipo I/metabolismo , Laminina/metabolismo , Subluxação do Cristalino/cirurgia , Subluxação do Cristalino/diagnóstico , Subluxação do Cristalino/metabolismo , Idoso de 80 Anos ou mais , Facoemulsificação , Síndrome de Exfoliação/metabolismo , Opacificação da Cápsula/metabolismo , Técnicas Imunoenzimáticas , Síndrome , Implante de Lente Intraocular , AdultoRESUMO
BACKGROUND: Posterior capsular opacification (PCO) is the main reason affecting the long-term postoperative result of cataract patient, and it is well accepted that fibrotic PCO is driven by transforming growth factor beta (TGFß) signaling. Ferroptosis, closely related to various ocular diseases, but has not been explored in PCO. METHODS: RNA sequencing (RNA-seq) was performed on both TGF-ß2 treated and untreated primary lens epithelial cells (pLECs). Differentially expressed genes (DEGs) associated with ferroptosis were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to investigate their biological function. Additionally, protein-to-protein interactions among selected ferroptosis-related genes by PPI network and the top 10 genes with the highest score (MCC algorithm) were selected as the hub genes. The top 20 genes with significant fold change values were validated using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Our analysis revealed 1253 DEGs between TGF-ß2 treated and untreated pLECs, uncovering 38 ferroptosis-related genes between two groups. Among these 38 ferroptosis-related genes,the most prominent GO enrichment analysis process involved in the response to oxidative stress (BPs), apical part of cell (CCs),antioxidant activity (MFs). KEGG were mainly concentrated in fluid shear stress and atherosclerosis, IL-17 and TNF signaling pathways, and validation of top 20 genes with significant fold change value were consistent with RNA-seq. CONCLUSIONS: Our RNA-Seq data identified 38 ferroptosis-related genes in TGF-ß2 treated and untreated pLECs, which is the first observation of ferroptosis related genes in primary human lens epithelial cells under TGF-ß2 stimulation.
Assuntos
Opacificação da Cápsula , Ferroptose , Humanos , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta2/farmacologia , Transcriptoma , Transição Epitelial-Mesenquimal/genética , Ferroptose/genética , Western Blotting , Opacificação da Cápsula/genética , Opacificação da Cápsula/metabolismo , Células Epiteliais/metabolismoRESUMO
Posterior capsule opacification (PCO) is the most common postoperative complication of cataract surgery. Transforming growth factor-ß (TGF-ß) is related to epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) that is proven to induce PCO formation in clinical and experimental studies. In this study, CRISPR sequences targeting exon of TGF-ßRII were knocked out with lentiviral transfection in LECs. Rabbits' PCO model was established and recombinant adeno-associated virus (AAV) for transferring the gRNA of TGF ßRII were intravitreally injected. SgRNA inhibited TGF-ßRII expression and human LECs proliferation. In TGF-ßRII knockout group, LECs motility and migration were suppressed, N-cadherin and vimentin expressions were significantly decreased, whereas E-cadherin was increased. The animal model showed that TGF-ßRII knockout in vivo was effective in suppressing PCO. The current study suggested that the CRISPR/Cas9 endonuclease system could suppress TGF-ßRII secretion, which participates in the EMT procedure of LECs in vitro and PCO in vivo. These findings might provide a new gene-editing approach and insight into a novel therapeutic strategy for PCO.
Assuntos
Opacificação da Cápsula , Cristalino , Animais , Humanos , Coelhos , Opacificação da Cápsula/genética , Opacificação da Cápsula/metabolismo , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , Cristalino/metabolismo , Células Epiteliais , Transição Epitelial-Mesenquimal/genética , Epitélio/metabolismo , Movimento Celular , Proliferação de CélulasRESUMO
Fibrotic cataract, including anterior subcapsular cataract (ASC) and posterior capsule opacification, always lead to visual impairment. Epithelial-mesenchymal transition (EMT) is a well-known event that causes phenotypic alterations in lens epithelial cells (LECs) during lens fibrosis. Accumulating studies have demonstrated that microRNAs are important regulators of EMT and fibrosis. However, the evidence explaining how microRNAs modulate the behavior and alter the cellular phenotypes of the lens epithelium in fibrotic cataract is insufficient. In this study, we found that hsa-let-7c-3p is downregulated in LECs in human ASC in vivo as well as in TGFß2-induced EMT in vitro, indicating that hsa-let-7c-3p may participate in modulating the profibrotic processes in the lens. We then demonstrated that overexpression of hsa-let-7c-3p markedly suppressed human LEC proliferation and migration and attenuated TGFß2-induced EMT and injury-induced ASC in a mouse model. In addition, hsa-let-7c-3p mediated lens fibrosis by directly targeting the CDH11 gene, which encodes cadherin-11 protein, an important mediator in the EMT signaling pathway. It decreased cadherin-11 protein expression at the posttranscriptional level but not at the transcriptional level by binding to a specific site in the 3-untranslated region (3'-UTR) of CDH11 mRNA. Moreover, blockade of cadherin-11 expression with a specific short hairpin RNA reversed TGFß2-induced EMT in LECs in vitro. Collectively, these data demonstrated that hsa-let-7c-3p plays a clear role in attenuating ASC development and may be a novel candidate therapeutic for halting fibrosis and maintaining vision.
Assuntos
Caderinas , Opacificação da Cápsula , Catarata , Cristalino , MicroRNAs , Animais , Humanos , Camundongos , Opacificação da Cápsula/genética , Opacificação da Cápsula/metabolismo , Catarata/genética , Catarata/metabolismo , Catarata/patologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Fibrose , Cristalino/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Glutaredoxin 2 (Grx2), a mitochondrial glutathione-dependent oxidoreductase, is crucial for maintaining redox homeostasis and cellular functions in the lens. The oxidative stress-induced epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is related to posterior capsule opacification. In this study, we investigated the effects of Grx2 on oxidative stress-induced EMT in LECs during posterior capsule opacification. We found that Grx2 expression was substantially decreased during the EMT of LECs and in a mouse model of cataract surgery. Deletion of Grx2 aggravated the generation of reactive oxygen species, including those that are mitochondria-derived, and promoted the proliferation and EMT of the LECs. This was reversed by Grx2 overexpression. In vivo, proteomic liquid chromatography-mass spectrometry analysis showed that integrin-linked kinase (ILK) was significantly upregulated in the lens posterior capsule of a Grx2 knockout (KO) mouse model. Compared with that of the wild-type group, the expression of ILK and EMT markers was increased in the Grx2 KO group which was reversed in the Grx2 knock-in group. Inhibition of ILK partially blocked Grx2 knockdown-induced EMT and prevented the increased phosphorylation of Akt and GSK-3ß and the nuclear translocation of ß-catenin in the Grx2 KO group. Finally, inhibition of the Wnt/ß-catenin pathway partially blocked the Grx2 knockdown-induced EMT. In conclusion, we demonstrated that Grx2 protects LECs from oxidative stress-related EMT by regulating the ILK/Akt/GSK-3ß axis.
Assuntos
Opacificação da Cápsula , Cristalino , Animais , Camundongos , beta Catenina/metabolismo , Opacificação da Cápsula/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutarredoxinas/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Cristalino/metabolismo , Camundongos Knockout , Estresse Oxidativo , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Regulação para CimaRESUMO
Herein, we review a unique and versatile lineage composed of Myo/Nog cells that may be beneficial or detrimental depending on their environment and nature of the pathological stimuli they are exposed to. While we will focus on the lens, related Myo/Nog cell behaviors and functions in other tissues are integrated into the narrative of our research that spans over three decades, examines multiple species and progresses from early stages of embryonic development to aging adults. Myo/Nog cells were discovered in the embryonic epiblast by their co-expression of the skeletal muscle-specific transcription factor MyoD, the bone morphogenetic protein inhibitor Noggin and brain-specific angiogenesis inhibitor 1. They were tracked from the epiblast into the developing lens, revealing heterogeneity of cell types within this structure. Depletion of Myo/Nog cells in the epiblast results in eye malformations arising from the absence of Noggin. In the adult lens, Myo/Nog cells are the source of myofibroblasts whose contractions produce wrinkles in the capsule. Eliminating this population within the rabbit lens during cataract surgery reduces posterior capsule opacification to below clinically significant levels. Parallels are drawn between the therapeutic potential of targeting Myo/Nog cells to prevent fibrotic disease in the lens and other ocular tissues.
Assuntos
Opacificação da Cápsula , Cristalino , Animais , Coelhos , Cristalino/metabolismo , Opacificação da Cápsula/metabolismo , Músculo Esquelético/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regulação da Expressão GênicaRESUMO
Purpose: TGFß-induced epithelial-to-myofibroblast transition (EMyT) of lens cells has been linked to the most common vision-disrupting complication of cataract surgery-namely, posterior capsule opacification (PCO; secondary cataract). Although inhibitors of the ErbB family of receptor tyrosine kinases have been shown to block some PCO-associated processes in model systems, our knowledge of ErbB signaling in the lens is very limited. Here, we investigate the expression of ErbBs and their ligands in primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) and how TGFß affects ErbB function. Methods: DCDMLs were analyzed by immunofluorescence microscopy and Western blotting under basal and profibrotic conditions. Results: Small-molecule ErbB kinase blockers, including the human therapeutic lapatinib, selectively inhibit TGFß-induced EMyT of DCDMLs. Lens cells constitutively express ErbB1 (EGFR), ErbB2, and ErbB4 protein on the plasma membrane and release into the medium ErbB-activating ligand. Culturing DCDMLs with TGFß increases soluble bioactive ErbB ligand and markedly alters ErbBs, reducing total and cell surface ErbB2 and ErbB4 while increasing ErbB1 expression and homodimer formation. Similar, TGFß-dependent changes in relative ErbB expression are induced when lens cells are exposed to the profibrotic substrate fibronectin. A single, 1-hour treatment with lapatinib inhibits EMyT in DCDMLs assessed 6 days later. Short-term exposure to lower doses of lapatinib is also capable of eliciting a durable response when combined with suboptimal levels of a mechanistically distinct multikinase inhibitor. Conclusions: Our findings support ErbB1 as a therapeutic target for fibrotic PCO, which could be leveraged to pharmaceutically preserve the vision of millions of patients with cataracts.
Assuntos
Opacificação da Cápsula , Catarata , Humanos , Opacificação da Cápsula/metabolismo , Lapatinib/metabolismo , Ligantes , Catarata/etiologia , Catarata/metabolismo , Células Epiteliais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , FibroseRESUMO
PURPOSE: Posterior capsule opacification (PCO) is the most common postoperative complication after cataract surgery and cannot yet be eliminated. Here, we investigated the inhibitory effects of telomerase reverse transcriptase (TERT) gene silencing on PCO in a rabbit model. METHODS: After rabbit lens epithelial cells (LECs) were treated with adenovirus containing short hairpin RNAs (shRNA) targeting TERT (shTERT group), adenovirus containing scramble nonsense control shRNA (shNC group) or PBS (control group), quantitative real-time polymerase chain reaction and Western blotting were used to measure the expression levels of TERT, and a scratch assay was performed to assess the LEC migration. New Zealand white rabbits underwent sham cataract surgery followed by an injection of adenovirus carrying shTERT into their capsule bag. The intraocular pressure and anterior segment inflammation were evaluated on certain days, and EMT markers (α-SMA and E-cadherin) were evaluated by Western blotting and immunofluorescence. The telomerase activity of the capsule bag was detected by ELISA. At 28 d postoperatively, hematoxylin and eosin staining of the cornea and iris and electron microscopy of the posterior capsule were performed. RESULTS: Application of shTERT to LECs downregulated the expression levels of TERT mRNA and protein. The scratch assay results showed a decrease in the migration of LECs in the shTERT group. In vivo, shTERT decreased PCO formation after cataract surgery in rabbits and downregulated the expression of EMT markers, as determined by Western blotting and immunofluorescence. In addition, telomerase activity was suppressed in the capsule bag. Despite slight inflammation in the iris, histologic results revealed no toxic effects in the cornea and iris. CONCLUSION: TERT silencing effectively reduces the migration and proliferation of LECs and the formation of PCO. Our findings suggest that TERT silencing may be a potential preventive strategy for PCO.
Assuntos
Opacificação da Cápsula , Catarata , Telomerase , Coelhos , Animais , Opacificação da Cápsula/genética , Opacificação da Cápsula/prevenção & controle , Opacificação da Cápsula/metabolismo , RNA Interferente Pequeno/genética , Adenoviridae/genética , Telomerase/genética , Telomerase/metabolismo , Inflamação/metabolismo , Células Epiteliais/metabolismo , Catarata/metabolismoRESUMO
PURPOSE: Posterior capsular opacification is the most common complication after cataract surgery. Abnormal proliferation, migration, epithelial-mesenchymal transition, and extracellular matrix synthesis of residual lens epithelial cells are considered to be the main pathogenic mechanisms. Hepatocyte nuclear factor 4α has been reported to regulate epithelial-mesenchymal transition in different tumors. Our objective was to investigate the role and mechanism of hepatocyte nuclear factor 4α in posterior capsular opacification. METHODS: Hepatocyte nuclear factor 4α expression was tested in posterior capsular opacification rat lens capsules and cell models. Hepatocyte nuclear factor 4α was knocked down using small hairpin RNA. Cell viability was measured by Cell Counting Kit-8 assay. Cell migration ability was evaluated by wound healing and Transwell assays. Epithelial-mesenchymal transition markers were detected by Western blotting. Transcriptome sequencing was used to screen for downstream effectors of hepatocyte nuclear factor 4α. Chromatin immunoprecipitation and a dual luciferase reporter assay were used to determine the binding of hepatocyte nuclear factor 4α to the MMP2 promoter region. RESULTS: Hepatocyte nuclear factor 4α was downregulated in posterior capsular opacification tissue and cell models. In vitro studies showed that hepatocyte nuclear factor 4α deletion facilitated cell proliferation, migration, and epithelial-mesenchymal transition protein marker expression in lens epithelial cells. Hepatocyte nuclear factor 4α knockdown promoted epithelial-mesenchymal transition and migration of lens epithelial cells via MMP2. Mechanistically, hepatocyte nuclear factor 4α decreased MMP2 expression by binding to the MMP2 promoter region. Hepatocyte nuclear factor 4α deletion also promoted epithelial-mesenchymal transition in rat lens capsules. CONCLUSIONS: We demonstrated that hepatocyte nuclear factor 4α inhibited epithelial-mesenchymal transition of lens epithelial cells by directly binding to the MMP2 promoter region and inhibiting the expression of MMP2, thus leading to retardation of posterior capsular opacification formation and development, suggesting that hepatocyte nuclear factor 4α is a potential therapeutic target for posterior capsular opacification.
Assuntos
Opacificação da Cápsula , Fator 4 Nuclear de Hepatócito , Cápsula do Cristalino , Cristalino , Metaloproteinase 2 da Matriz , Animais , Ratos , Opacificação da Cápsula/metabolismo , Cápsulas/metabolismo , Movimento Celular , Proliferação de Células , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Cápsula do Cristalino/patologia , Cristalino/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismoRESUMO
Fibroblast growth factor (FGF) and transforming growth factor-beta (TGF-ß) can regulate and/or dysregulate lens epithelial cell (LEC) behaviour, including proliferation, fibre differentiation, and epithelial-mesenchymal transition (EMT). Earlier studies have investigated the crosstalk between FGF and TGF-ß in dictating lens cell fate, that appears to be dose dependent. Here, we tested the hypothesis that a fibre-differentiating dose of FGF differentially regulates the behaviour of lens epithelial cells undergoing TGF-ß-induced EMT. Postnatal 21-day-old rat lens epithelial explants were treated with a fibre-differentiating dose of FGF-2 (200 ng/mL) and/or TGF-ß2 (50 pg/mL) over a 7-day culture period. We compared central LECs (CLECs) and peripheral LECs (PLECs) using immunolabelling for changes in markers for EMT (α-SMA), lens fibre differentiation (ß-crystallin), epithelial cell adhesion (ß-catenin), and the cytoskeleton (alpha-tropomyosin), as well as Smad2/3- and MAPK/ERK1/2-signalling. Lens epithelial explants cotreated with FGF-2 and TGF-ß2 exhibited a differential response, with CLECs undergoing EMT while PLECs favoured more of a lens fibre differentiation response, compared to the TGF-ß-only-treated explants where all cells in the explants underwent EMT. The CLECs cotreated with FGF and TGF-ß immunolabelled for α-SMA, with minimal ß-crystallin, whereas the PLECs demonstrated strong ß-crystallin reactivity and little α-SMA. Interestingly, compared to the TGF-ß-only-treated explants, α-SMA was significantly decreased in the CLECs cotreated with FGF/TGF-ß. Smad-dependent and independent signalling was increased in the FGF-2/TGF-ß2 co-treated CLECs, that had a heightened number of cells with nuclear localisation of Smad2/3 compared to the PLECs, that in contrast had more pronounced ERK1/2-signalling over Smad2/3 activation. The current study has confirmed that FGF-2 is influential in differentially regulating the behaviour of LECs during TGF-ß-induced EMT, leading to a heterogenous cell population, typical of that observed in the development of post-surgical, posterior capsular opacification (PCO). This highlights the cooperative relationship between FGF and TGF-ß leading to lens pathology, providing a different perspective when considering preventative measures for controlling PCO.
Assuntos
Opacificação da Cápsula , Fator de Crescimento Transformador beta2 , Ratos , Animais , Fator de Crescimento Transformador beta2/farmacologia , Fator de Crescimento Transformador beta2/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Transição Epitelial-Mesenquimal , Opacificação da Cápsula/metabolismo , Células Epiteliais/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/metabolismo , beta-Cristalinas/metabolismoRESUMO
Purpose: Posterior capsular opacification (PCO) is the most common complication postcataract surgery, and its underlying mechanisms involve epithelial-mesenchymal transition (EMT) of remnant lens epithelial cells (LECs) in response to drastic changes in stimuli in the intraocular environment, such as oxidative stress and growth factors. Wnt/ß-catenin signaling is a major pathway mediating oxidative stress-induced EMT in LECs, but its interplay with other transduction pathways remains little known in the development of PCO. ERK1/2 signaling is the downstream component of a phosphorelay pathway in response to extracellular stimuli (e.g., reactive oxygen species), and its activation regulates multiple cellular processes, including proliferation and EMT. Thus, this study aimed to investigate how ERK1/2 signaling and Wnt/ß-catenin pathway crosstalk in oxidative stress-induced EMT in LECs. Methods: Hydrogen peroxide (H2O2) at 50 µM treatment for 48 h was used to establish a moderate oxidative stress-induced EMT model in LECs. ERK1/2 signaling was inhibited using MEK1/2 inhibitor U0126 at 20 µM. Western blotting was used to quantify protein expression of various biomarkers of EMT and phosphorylated components in ERK1/2 and Wnt/ß-catenin signaling. LEC proliferation was determined using an EdU staining assay and expression of proliferating cellular nuclear antigen (PCNA). Subcellular localization of biomarker proteins was visualized with immunofluorescent staining. Results: Under the moderate level of H2O2-induced EMT in LECs, ERK1/2 signaling was activated, as evidenced by a marked increase in the ratio of phosphorylated ERK1/2 to total ERK1/2 at early (i.e., 5-15 min) and late time points (i.e., 12 h); the canonical Wnt/ß-catenin pathway was activated by H2O2 at 48 h. LECs exposed to H2O2 exhibited hyperproliferation and EMT; however, these were restored by inhibition of ERK1/2 signaling demonstrated by reduced DNA synthesis and PCNA expression for cellular proliferation and altered expression of various EMT protein markers, including E-cadherin, α-SMA, and vimentin. More importantly, inhibition of ERK1/2 signaling reduced ß-catenin accumulation in the activated Wnt/ß-catenin signaling cascade. Specifically, there was significant downregulation in the phosphorylation level of LRP6 at Ser 1490 and GSK-3ß at Ser 9, the key coreceptor of Wnt and regulator of ß-catenin, respectively. Conclusions: ERK1/2 signaling plays a crucial role in the moderate level of oxidative stress-induced EMT in LECs. Pharmacologically blocking ERK1/2 signaling significantly inhibited LEC proliferation and EMT. Mechanistically, ERK1/2 signaling regulated Wnt/ß-catenin cascade by phosphorylating Wnt coreceptor LRP6 at Ser 1490 in the plasma membrane. These results shed light on a potential molecular switch of ERK1/2 and Wnt/ß-catenin crosstalk underlying the development of PCO.
Assuntos
Opacificação da Cápsula , Sistema de Sinalização das MAP Quinases , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , beta Catenina/metabolismo , Miofibroblastos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Peróxido de Hidrogênio/farmacologia , Opacificação da Cápsula/metabolismo , Via de Sinalização Wnt , Células Epiteliais/metabolismo , Estresse Oxidativo , Transição Epitelial-Mesenquimal/genéticaRESUMO
Cataracts are treated by lens fiber cell removal followed by intraocular lens (IOL) implantation into the lens capsule. While effective, this procedure leaves behind numerous lens epithelial cells (LECs) which undergo a wound healing response that frequently leads to posterior capsular opacification (PCO). In order to elucidate the acute response of LECs to lens fiber cell removal which models cataract surgery (post cataract surgery, PCS), RNA-seq was conducted on LECs derived from wild type mice at 0 and 6 h PCS. This analysis found that LECs upregulate the expression of numerous proinflammatory cytokines and profibrotic regulators by 6 h PCS suggesting rapid priming of pathways leading to inflammation and fibrosis PCS. LECs also highly upregulate the expression of numerous immediate early transcription factors (IETFs) by 6 h PCS and immunolocalization found elevated levels of these proteins by 3 h PCS, and this was preceded by the phosphorylation of ERK1/2 in injured LECs. Egr1 and FosB were among the highest expressed of these factors and qRT-PCR revealed that they also upregulate in explanted mouse lens epithelia suggesting potential roles in the LEC injury response. Analysis of lenses lacking either Egr1 or FosB revealed that both genes may regulate a portion of the acute LEC injury response, although neither gene was essential for expression of either proinflammatory or fibrotic markers at later times PCS suggesting that IETFs may work in concert to mediate the LEC injury response following cataract surgery.
Assuntos
Opacificação da Cápsula , Extração de Catarata , Traumatismos Oculares , Cápsula do Cristalino , Cristalino , Camundongos , Animais , Cápsula do Cristalino/metabolismo , Cápsula do Cristalino/patologia , Cristalino/metabolismo , Células Epiteliais/metabolismo , Opacificação da Cápsula/metabolismo , Traumatismos Oculares/metabolismo , Fatores de Transcrição/metabolismo , FibroseRESUMO
Posterior capsular opacification (PCO) is the most common complication after cataract surgery, which is primarily caused by the proliferation of the residual lens epithelial cells (LECs) in the lens capsule. Previous studies have demonstrated that a drug-eluting intraocular lens (IOL), aimed to in situ eliminate LECs, are an effective and promising way to prevent PCO. However, because of the potential toxicities of the antiproliferative drugs to the adjacent tissues, the safety of such drug-eluting IOLs is still a highly important issue to be solved. In this investigation, a facile photodynamic coating-modified IOL was developed for effective and safer PCO prevention. An annular poly(lactide-co-glycolic acid) (PLGA) coating loaded with photosensitizer chlorin e6 (Ce6) was prepared by a spin-coating technique. The optical property investigations showed that the Ce6@PLGA coating was particularly suitable for the IOL surface modification. The in vitro cell culture investigation showed that Ce6@PLGA coating-modified IOLs effectively eliminated LECs when treated with light illumination, whereas it appeared to have good cytocompatibility without irradiation. The investigation of the cell elimination mechanism showed that the apoptosis of HLECs may be associated with the cytomembrane disruption induced by ROS, which is generated by the photodynamic coating during light illumination. The in vivo implantation experiments confirmed the desired PCO prevention effect, as well as the safety to and biocompatibility with the surrounding tissues. Thus, the facile Ce6@PLGA coating will provide an effective yet safe alternative of IOL surface modification for PCO prevention.
Assuntos
Opacificação da Cápsula , Cápsula do Cristalino , Cristalino , Lentes Intraoculares , Humanos , Proliferação de Células , Opacificação da Cápsula/metabolismo , Cápsula do Cristalino/metabolismo , Cristalino/metabolismoRESUMO
Aim: To formulate an injectable thermosensitive micelle-hydrogel hybrid system loaded with celastrol (celastrol-loaded micelle hydrogel: CMG) to prevent posterior capsule opacification (PCO). Materials & methods: Celastrol-loaded micelles were embedded in a thermosensitive hydrogel matrix to enable controlled on-demand celastrol delivery into the residual capsule. The efficacy and mechanisms of the system for eliminating PCO were evaluated in rabbits. Results: Celastrol-loaded micelles inhibited the migration and proliferation of lens epithelial cells induced by TGF-ß1. Celastrol prevents epithelial-mesenchymal transition in lens epithelial cells induced by TGF-ß1 through the TGF-ß1/Smad2/3/TEAD1 signaling pathway. In vivo efficiency evaluations showed that CMG demonstrated an excellent inhibitory effect on PCO in rabbits and had no obvious tissue toxicity. Conclusion: Injectable CMG may represent a promising ophthalmic platform for preventing PCO. This versatile injectable micelle-hydrogel hybrid represents a clinically relevant platform to achieve localized therapy and controlled release of drugs in other disease therapies.
Assuntos
Opacificação da Cápsula , Animais , Coelhos , Opacificação da Cápsula/tratamento farmacológico , Opacificação da Cápsula/prevenção & controle , Opacificação da Cápsula/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Micelas , Hidrogéis/farmacologia , Nanomedicina , Transição Epitelial-Mesenquimal , Células Epiteliais/metabolismoRESUMO
Growth factor-induced migration of lens epithelial cell (LEC) toward the posterior of lens capsule bag and their epithelial-mesenchymal transition (EMT) is the key process involved in the pathogenesis of posterior capsular opacification (PCO). Silibinin, a natural flavonolignan, confers therapeutic effects to different cells by regulation of signalling pathways; however, its role in the prevention of migration and EMT of LECs is yet to be analysed. In this study, the inhibitory capabilities of silibinin on migration and EMT were analysed in response to TGFß2 stimulation in HLE B-3 cells. The anti-migratory effect of silibinin was analysed using wound healing assay. Transcriptional and translational expression of genes related to LEC migration, EMT, and transcription factors related to EMT were studied by quantitative real-time PCR and Western blotting. Immunofluorescence analysis was utilized to study the localization of fibronectin. Silibinin reduced the viability of LECs in a concentration-dependent manner and inhibited the wound healing capacity of LECs induced by TGFß2. Silibinin also suppressed alteration in the EMT-related markers such as cytoskeletal proteins, cell adhesion markers, extracellular matrix molecules, and transcription factors. Analysis of downstream signalling revealed that treatment with silibinin decreased phosphorylated Akt (Ser473, Thr308), PDK1 (Ser241), PTEN (Ser380), c-Raf (Ser259), and GSK3ß (Ser9) in TGFß-stimulated cells. The effect of silibinin treatment on phosphorylated Akt resembled that of the PI3K inhibitor LY294002. Our results suggest that silibinin can suppress LEC migration and EMT, which involves the inactivation of the PI3K-Akt signalling pathway. Silibinin might be a good candidate for PCO prevention; however, functional evaluation of silibinin using in vivo models is a pre-requisite.
Assuntos
Opacificação da Cápsula , Flavonolignanos , Cristalino , Opacificação da Cápsula/metabolismo , Movimento Celular , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Fibronectinas/metabolismo , Flavonolignanos/metabolismo , Flavonolignanos/farmacologia , Glicogênio Sintase Quinase 3 beta , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Silibina/metabolismo , Silibina/farmacologia , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta2/farmacologiaRESUMO
The integrity of lens epithelial cells (LECs) lays the foundation for lens function and transparency. By contrast, epithelial-mesenchymal transition (EMT) of LECs leads to lens fibrosis, such as anterior subcapsular cataracts (ASC) and fibrotic forms of posterior capsule opacification (PCO). However, the underlying mechanisms remain unclear. Here, we aimed to explore the role of long non-coding RNA (lncRNA) H19 in regulating TGF-ß2-induced EMT during lens fibrosis, revealing a novel lncRNA-based regulatory mechanism. In this work, we identified that lncRNA H19 was highly expressed in LECs, but downregulated by exposure to TGF-ß2. In both human lens epithelial explants and SRA01/04 cells, knockdown of H19 aggravated TGF-ß2-induced EMT, while overexpressing H19 partially reversed EMT and restored lens epithelial phenotypes. Semi-in vivo whole lens culture and H19 knockout mice demonstrated the indispensable role of H19 in sustaining lens clarity through maintaining LEC features. Bioinformatic analyses further implied a potential H19-centered regulatory mechanism via Smad-dependent pathways, confirmed by in vitro experiments. In conclusion, we uncovered a novel role of H19 in inhibiting TGF-ß2-induced EMT of the lens by suppressing Smad-dependent signaling, providing potential therapeutic targets for treating lens fibrosis.
Assuntos
Opacificação da Cápsula , RNA Longo não Codificante , Animais , Opacificação da Cápsula/genética , Opacificação da Cápsula/metabolismo , Células Epiteliais/metabolismo , Fibrose , Humanos , Camundongos , Fenótipo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fator de Crescimento Transformador beta2/metabolismoRESUMO
BACKGROUND: Posterior capsule opacification (PCO) is the most common complication after cataract surgery, in which increased levels of transforming growth factor-beta 2 (TGF-ß2) accelerate PCO formation; however, the pathological mechanisms are not fully understood. This study aims to explore the regulation mechanism of TGF-ß2 in PCO formation via its autophagic functions. METHODS: The autophagic effect of TGF-ß2 was detected by transmission electron microscopy (TEM), Western blotting, and immunofluorescence analysis. The association between autophagy and the epithelial-mesenchymal transition (EMT) was evaluated by qPCR and Western blotting. The transcriptome analysis was used to uncover the molecular mechanism of TGF-ß2-induced PCO formation. RESULTS: TGF-ß2 specifically promotes autophagy flux in human lens epithelial cells. The activation of autophagy by rapamycin can promote EMT marker synthesis and improve cell migration. However, the inhibition of autophagy by 3-MA attenuates EMT. To uncover the molecular mechanisms, we performed RNA sequencing and found that TGF-ß2 elevated tumor protein p53-inducible nuclear protein2 (TP53INP2) expression, which was accompanied by a nuclear-to-cytoplasm translocation. Moreover, the knockdown of TP53INP2 blocked the TGF-ß2-induced autophagy and EMT processes, revealing that TP53INP2 plays an important role in TGF-ß2-induced autophagy during EMT. CONCLUSIONS: Taken together, the results of this study suggested that TP53INP2 was a novel regulator of PCO development by TGF-ß2, and notably, TP53INP2, may be a potential target for the pharmacological treatment of PCO.
Assuntos
Opacificação da Cápsula , Proteínas Nucleares/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Autofagia , Opacificação da Cápsula/metabolismo , Transição Epitelial-Mesenquimal/genética , Humanos , Proteína Supressora de Tumor p53RESUMO
The abnormal proliferation, migration, and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs) are the main reasons for vision loss caused by posterior capsular opacification (PCO) after cataract surgery. Insulin-like growth factor-1 (IGF-1) was found to be associated with the pathogenesis of cataracts, but its biological role in PCO is poorly understood. In the present study, IGF-1 overexpression facilitated the proliferation, migration, and EMT, whereas knockdown of IGF-1 markedly suppressed the proliferation, migration, and TGF-ß2-induced EMT of LECs. Additionally, to evaluate valuable microRNAs (miRNAs) which target IGF-1 to modulate LEC-EMT, we predicted miR-3666 might regulate IGF-1 by binding its 3'UTR according to the bioinformatics database. Furthermore, we verified that miR-3666 directly targeted IGF-1 by luciferase reporter assay. By using miR-3666 mimics, cell proliferation, migration, and invasion were suppressed, while being enhanced by the reduction of miR-3666. Knockout of IGF1 reverses the effect of the miR-3666 inhibitor on the malignant behavior of LECs. These results indicate the role of miR-3666/IGF-1 in LEC-EMT that offers new strategies for the therapy and prevention of PCO.
Assuntos
Opacificação da Cápsula , Fator de Crescimento Insulin-Like I , Cristalino , MicroRNAs , Opacificação da Cápsula/genética , Opacificação da Cápsula/metabolismo , Opacificação da Cápsula/patologia , Movimento Celular/genética , Proliferação de Células , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/fisiologia , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Cristalino/metabolismo , Cristalino/patologia , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Posterior capsule opacification (PCO) is a frequent complication after cataract surgery, and advanced PCO requires YAG laser (Nd: YAG) capsulotomy, which often gives rise to more complications. Lens epithelial cell (LEC) proliferation and transformation (i.e., epithelial-mesenchymal transition (EMT)) are two critical elements in PCO initiation and progression pathogenesis. While PCO marginally impacts aged cataract surgery patients, PCO incidences are exceptionally high in infants and children undergoing cataract surgery. The gene expression of lens epithelial cell aging and its role in the discrepancy of PCO prevalence between young and older people have not been fully studied. Here, we conducted a comprehensive differentially expressed gene (DEG) analysis of a cell aging model by comparing the early and late passage FHL124 lens epithelial cells (LECs). In vitro, TGFß2, cell treatment, and in vivo mouse cataract surgical models were used to validate our findings. We found that aged LECs decelerated rates of cell proliferation accompanied by dysregulation of cellular immune response and cell stress response. Surprisingly, we found that LECs systematically downregulated epithelial-mesenchymal transition (EMT)-promoting genes. The protein expression of several EMT hallmark genes, e.g., fibronectin, αSMA, and cadherin 11, were gradually decreased during LECs aging. We then confirmed these findings in vitro and found that aged LECs markedly alleviated TGFß2-mediated EMT. Importantly, we explicitly confirmed the in vitro findings from the in vivo mouse cataract surgery studies. We propose that both the high proliferation rate and EMT-enriched young LECs phenotypic characteristics contribute to unusually high PCO incidence in infants and children.