Epithelial-mesenchymal Transition Of Lens Epithelial Cells Research Articles

Binding with HSP90β, cimifugin ameliorates fibrotic cataracts in vitro and in vivo by inhibiting TGFβ signaling pathways

Fibrotic cataracts, the most frequent complications after phacoemulsification, cannot be cured by drugs in clinic. The primary mechanism underlying the disease is the epithelial-mesenchymal transition (EMT). Cimifugin is a natural monomer component of traditional Chinese medicines. Previous researches have demonstrated the effect of cimifugin inhibiting EMT in the lung. The purpose of this work is to evaluate the impact of cimifugin on EMT in the lens and elucidate its precise mechanism. The pathogenesis of fibrotic cataracts was simulated using TGFβ2-induced cell model of EMT and the injury-induced anterior subcapsular cataract animal model. Through H&E staining and immunofluorescence of mice eyeballs, we discovered that cimifugin can inhibit the expansion of fibrotic lesions in vivo. Furthermore, at mRNA and protein levels, we confirmed that cimifugin can allay EMT of lens epithelial cells (LECs) in vitro and in vivo. Additionally, the inhibition of cimifugin on the activation of TGFβ-related signaling pathways was certified by immunoblot. HSP90β, the target of cimifugin, was predicted by network pharmacology and verified by drug affinity responsive target stability, the cellular thermal shift assay, and microscale thermophoresis. Moreover, co-immunoprecipitation revealed the interaction between HSP90β and TGFβ receptor (TGFβR) II. Together, our findings showed that by weakening the binding of HSP90β and TGFβRII, cimifugin suppressed the TGFβ signaling pathways to alleviate fibrotic cataracts. Cimifugin is a promising medication for the treatment of fibrotic cataracts.

Indomethacin and bromfenac may be effective in the prevention of posterior capsule opacification

Posterior capsule opacification (PCO) is the most common long‐term postoperative complication of cataract surgery, leading to secondary vision loss. Optimized intraocular lens (IOL) structure and appropriate pharmacological intervention can provide physical barriers and biological inhibition, respectively, to block the migration, proliferation, and epithelial‐mesenchymal transition (EMT) of lens epithelial cells (LECs) for PCO prophylaxis. We demonstrated that non‐steroidal anti‐inflammatory drugs (NSAIDs) are effective in preventing posterior capsule opacification. Indomethacin (INDOM) not only suppresses cell migration and down‐regulates the expression of cyclooxygenase‐2 (COX‐2) and EMT markers, including alpha‐smooth muscle actin (α‐SMA) and cyclin D1, but also promotes autophagy activation in LECs. Autophagy was also verified as a potential therapeutic target for down‐regulating EMT in LECs. Bromfenac effectively inhibits cell migration, overexpression of EMT markers such as fibronectin (FN), matrix metalloproteinase 2 (MMP2), α‐smooth muscle actin (α‐SMA), and transcription factor Snail, and extracellular signal‐regulated kinase (ERK)/glycogen synthase kinase‐3β (GSK‐3β) signalling induced by transforming growth factor‐β2 (TGF‐β2) in vitro. Based on ultrasonic spray technology, we developed a drug‐eluting intraocular lens (IOL) using poly (lactic‐co‐glycolic acid) (PLGA) with sustained release ability of indomethacin/bromfenac for PCO prevention. The novel IOL, serving as a drug delivery platform, can carry an adjustable dose of drugs with sustained release ability for more than 28 days. In the rabbit PCO model, the novel IOL showed excellent anti‐inflammatory and anti‐PCO effects. In summary, indomethacin and bromfenac are effective pharmacological interventions for PCO prophylaxis, and the novel IOL we developed prevented PCO in vivo, providing a promising approach for PCO prophylaxis in clinical applications.

Attenuation of epithelial-mesenchymal transition via SGLT2 inhibition and diabetic cataract suppression by dapagliflozin nanoparticles treatment

AimsChronic hyperglycemia triggers overproduction of AKR1B1 (aldo-keto reductase family 1 member B) and receptor for advanced glycation end product (RAGE), which causes epithelial-mesenchymal transition (EMT) in the lens epithelial cells (LECs) of diabetic mellitus (DM) cataracts. However, it is unclear whether EMT in LECs is related to abnormal increase of SGLT2. Sodium glucose cotransporter 2 (SGLT2) inhibitor, also known as dapagliflozin (Dapa) can be used to treat diabetes. Here, we examined how Dapa or nano eye-drops (DapaN) reduce EMT in LECs of DM cataracts. The nano eye-drop provides an ophthalmic treatment that suppressed diabetic cataract progression and improved potency with reduced side effects. Main methodsSD rats were injected with streptozocin (STZ) (65 mg/kg, ip), nano-Dapa drops (0.456 mg/10 ml/eye) or Dapa (1.2 mg/kg/day) treatment for 6–12 weeks. Immunofluorescence staining was used for protein quantification of RAGE, SGLT2, N-cadherin and E-cadherin in the LECs of rats. Key findingsIn this study, Dapa applies nanotechnology-based delivery system and it contains polyvinylpyrrolidone (PVP) and HPBCD. Dapa showed therapeutic effect on DM cataracts, wherein it targeted EMT biomarker, E-cadherin. The nano-Dapa drops or oral Dapa inhibited SGLT2, suppressed AKR1B1 expression, decreased AcSOD2- and RAGE-induced EMT in diabetic cataracts. Our findings suggest that nanotechnology-based Dapa eye drops (Dapa-PVP-HPBCD) can effectively improve solubility of Dapa in aqueous solution. SignificanceTaken together, results suggest that the SGLT2-mediated DM cataract therapy may involve the AKR1B1-RAGE-AcSOD2-EMT pathway. The nano eye drops and Dapa show potential beneficial effects for cataract prevention. This study conveys new insights into cataract treatment and supplementation of nano-Dapa drops shows promising result in preventing diabetic cataracts.

Glutaredoxin 2 protects lens epithelial cells from epithelial-mesenchymal transition by suppressing mitochondrial oxidative stress-related upregulation of integrin-linked kinase

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-3β and the nuclear translocation of β-catenin in the Grx2 KO group. Finally, inhibition of the Wnt/β-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-3β axis.

Role of Chondroitin Sulfate Proteoglycan 5 in Steroid-Induced Cataract.

Steroid-induced cataracts (SIC) are defined as cataracts associated with the administration of corticosteroids. Long-term glucocorticoid treatment for inflammatory diseases reportedly increases the risk of SIC, and steroids can induce cataracts by disrupting ocular growth factor balance or homeostasis. In this study, we verified the effect of chondroitin sulfate proteoglycan 5 (CSPG5) using dexamethasone (dexa)-treated human lens epithelial (HLE-B3) cells and the lens epithelium from the anterior capsule of SIC patients obtained during cataract surgery. CSPG5 expression increased in the lens epithelium of SIC patients. The downregulation of CSPG5 suppressed the dexa-induced epithelial-mesenchymal transition (EMT)-related protein expression and motility in HLE-B3 cells. The disruption of the transcription factors EZH2 and B-Myb downregulated CSPG5, dexa-induced fibronectin expression, and cell migration in HLE-B3 cells, reaffirming that CSPG5 expression regulates EMT in lens epithelial cells. Taken together, these results indicate that the steroid-induced effects on lens epithelial cells are mediated via alterations in CSPG5 expression. Therefore, our study emphasizes the potential of CSPG5 as a therapeutic target for the prevention and treatment of SIC.

Let-7c-3p suppresses lens epithelial-mesenchymal transition by inhibiting cadherin-11 expression in fibrotic cataract.

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 TGFβ2-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 TGFβ2-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 TGFβ2-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.

Prophylaxis of posterior capsule opacification through autophagy activation with indomethacin-eluting intraocular lens.

Posterior capsule opacification (PCO) is the most common long-term postoperative complication of cataract surgery, leading to secondary vision loss. Optimized intraocular lens (IOL) structure and appropriate pharmacological intervention, which provides physical barriers and biological inhibition, respectively, can block the migration, proliferation, and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) for PCO prophylaxis. Herein, a novel indomethacin-eluting IOL (INDOM-IOL) with an optimized sharper edge and a sustained drug release behavior was developed for PCO prevention. Indomethacin (INDOM), an ophthalmic non-steroidal anti-inflammatory drug (NSAID) used for postoperative ocular inflammation, was demonstrated to not only be able to suppress cell migration and down-regulate the expression of cyclooxygenase-2 (COX-2) and EMT markers, including alpha-smooth muscle actin (α-SMA) and cyclin D1, but also promote the autophagy activation in LECs. Additionally, autophagy was also verified to be a potential therapeutic target for the down-regulation of EMT in LECs. The novel IOL, serving as a drug delivery platform, could carry an adjustable dose of hydrophobic indomethacin with sustained drug release ability for more than 28 days. In the rabbit PCO model, the indomethacin-eluting IOL showed excellent anti-inflammatory and anti-PCO effects. In summary, indomethacin is an effective pharmacological intervention in PCO prophylaxis, and the novel IOL we developed prevented PCO in vivo under its sustained indomethacin release property, which provided a promising approach for PCO prophylaxis in clinical application.

BMP-4 and BMP-7 Inhibit EMT in a Model of Anterior Subcapsular Cataract in Part by Regulating the Notch Signaling Pathway.

The proliferation, migration, and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) are believed to be the pathological mechanisms underlying anterior subcapsular cataract (ASC). Bone morphogenetic proteins (BMPs) inhibit transforming growth factor-beta (TGF-β)-induced fibrosis in the lens. Herein, we aimed to further clarify the roles of BMP-4/BMP-7 in the progression and the underlying mechanisms of fibrotic cataract. BMP-4/BMP-7, TGF-β2, jagged-1 peptide, or DAPT were applied in a mouse injury-induced ASC model and in the human LEC cell line SRA01/04. The volume of opacity was examined by a slit lamp and determined by lens anterior capsule whole-mount immunofluorescence. Global gene expression changes were assessed by RNA sequencing, and the levels of individual mRNAs were validated by real-time PCR. Protein expression was determined by the Simple Western sample dilution buffer. Cell proliferation was examined by CCK8 and EdU assays, and cell migration was measured by Transwell and wound healing assays. Anterior chamber injection of BMP-4/BMP-7 significantly suppressed subcapsular opacification formation. RNA sequencing of the mouse ASC model identified the Notch pathway as a potential mechanism involved in BMP-mediated inhibition of ASC. Consistently, BMP-4/BMP-7 selectively suppressed Notch1 and Notch3 and their downstream genes, including Hes and Hey. BMP-4/BMP-7 or DAPT suppressed cell proliferation by inducing G1 cell cycle arrest. BMP-4/BMP-7 also inhibited TGF-β2-induced cell migration and EMT by modulating the Notch pathway. BMP-4/BMP-7 attenuated ASC by inhibiting proliferation, migration, and EMT of LECs via modulation of the Notch pathway, thereby providing a new avenue for ASC treatment.

Blue light attenuates TGF-β2-induced epithelial-mesenchymal transition in human lens epithelial cells via autophagy impairment

BackgroundPathogenesis of posterior capsular opacification (PCO) was related to pathological epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). It has been reported that blue light could have an effect on EMT. This study aims to elucidate the role and potential mechanism of autophagy in EMT after blue light exposure in LECs.MethodsHLE-B3 cells were treated with TGF-β2 with different concentration and time to induce EMT as a model of PCO in vitro. Cells were exposed to blue light with or without TGF-β2. The expression levels of EMT-associated markers were analyzed by qRT-PCR, western blotting and cell migration ability was determined by transwell migration assay and wound healing assay. The expressions of autophagy-related proteins were analyzed by western blotting, immunofluorescence and transmission electron microscopy. Rapamycin and chloroquine were utilized in cells for autophagy activation and inhibition.ResultsTGF-β2 induced autophagy activation during EMT progression in HLE-B3 cells in a dose- and time-dependent manner. Blue light exposure inhibited TGF-β2-induced EMT characterized by inhibited expression of EMT related markers and reduced migration capacity. Meanwhile, blue light exposure impaired autophagy activated by TGF-β2. Furthermore, Autophagy activation with rapamycin rescued EMT attenuated by blue light. Autophagy inhibition with chloroquine reduced TGF-β2-induced EMT in HLE-B3 cells.ConclusionBlue light exposure had inhibited effects on TGF-β2-induced EMT in LECs through autophagy impairment, which provides a new insight on prevention and treatment of PCO.

The interaction between autophagy and the epithelial-mesenchymal transition mediated by NICD/ULK1 is involved in the formation of diabetic cataracts

BackgroundCataracts are the leading cause of blindness and a common ocular complication of diabetes. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) and altered autophagic activity occur during the development of diabetic cataracts. The disturbed interaction of autophagy with EMT in LECs stimulated by high glucose levels may participate in cataract formation.MethodsA rat diabetic cataract model induced by streptozotocin (STZ) and human lens epithelial cells (HLE-B3) stimulated with a high glucose concentration were employed in the study. These models were treated with rapamycin (an inhibitor of mammalian target of rapamycin (mTOR)), and N-(N-[3,5-difluorophenacetyl]-1-alanyl)-S-phenylglycine t-butyl ester (DAPT, an inhibitor of γ-secretase) alone or in combination. Lens opacity was observed and photographed under a slit-lamp microscope. Histological changes in paraffin sections of lenses were detected under a light microscope after hematoxylin and eosin staining. Alterations of autophagosomes in LECs were counted and evaluated under a transmission electron microscope. The expression levels of proteins involved in the EMT, autophagy, and the signaling pathways in LECs were measured using Western blotting and immunofluorescence staining. Cell migration was determined by performing transwell and scratch wound assays. Coimmunoprecipitation (Co-IP) was performed to verify protein-protein interactions. Proteins were overexpressed in transfected cells to confirm their roles in the signaling pathways of interest.ResultsIn LECs, a high glucose concentration induces the EMT by activating Jagged1/Notch1/Notch intracellular domain (NICD)/Snail signaling and inhibits autophagy through the AKT/mTOR/unc 51-like kinase 1 (ULK1) signaling pathway in vivo and in vitro, resulting in diabetic cataracts. Enhanced autophagic activity induced by rapamycin suppressed the EMT by inducing Notch1 degradation by SQSTM1/p62 and microtubule-associated protein light chain 3 (LC3) in LECs, while inhibition of the Notch signaling pathway with DAPT not only prevented the EMT but also activated autophagy by decreasing the levels of NICD, which bound to ULK1, phosphorylated it, and then inhibited the initiation of autophagy.ConclusionsWe describe a new interaction of autophagy and the EMT involving NICD/ULK1 signaling, which mediates crosstalk between these two important events in the formation of diabetic cataracts. Activating autophagy and suppressing the EMT mutually promote each other, revealing a potential target and strategy for the prevention of diabetic cataracts.

Sprouty and Spred temporally regulate ERK1/2-signaling to suppress TGFβ-induced lens EMT

Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) principally contributes to the pathogenesis of fibrotic cataract. Sprouty (Spry) and Spred proteins are receptor tyrosine kinase (RTK) antagonists that can regulate RTK-mediated signaling pathways, such as the MAPK/ERK1/2-signaling pathway. The present study examines the ability of Spry and Spred to inhibit TGFβ-induced EMT in LECs. LECs explanted from postnatal-day-21 Wistar rats were transduced with adenoviral vectors coding for Spry1, Spry2 or Spred2, and subsequently treated with or without TGFβ2. Immunofluorescent labeling of explants for the epithelial membrane marker β-catenin, and the mesenchymal marker alpha-smooth muscle actin (α-sma), were used to characterize the progression of EMT. Western blotting was used to quantify levels of α-sma and ERK1/2-signaling. Overexpression of Spry or Spred in LECs was sufficient to suppress EMT in response to TGFβ, including a block to cell elongation, β-catenin delocalization and α-sma accumulation. Spry and Spred were also shown to significantly block ERK1/2 phosphorylation for up to 18 h of TGFβ treatment but did not impair the earlier activation of ERK1/2 at 20 min. These findings suggest that Spry and Spred may not directly impact ERK1/2-signaling activated by the serine/threonine kinase TGFβ receptor, but may selectively target later ERK1/2-signaling driven by downstream RTK-mediated signaling. Taken together, our data establish Spry and Spred antagonists as potent negative regulators of TGFβ-induced EMT that can regulate ERK1/2-signaling in a temporal manner. A greater understanding of how Spry and Spred regulate the complex signaling interactions that underlie TGFβ-induced EMT will be essential to facilitate the development of novel therapeutics for different pathologies driven by EMT, including fibrotic forms of cataract.

Paeoniflorin inhibits epithelial mesenchymal transformation and oxidative damage of lens epithelial cells in diabetic cataract via sirtuin 1 upregulation

ABSTRACT Paeoniflorin (Pae) has been reported to serve an important role in complications associated with diabetes. To the best of our knowledge, the role of Pae in diabetic cataracts has not yet been reported. Human lens epithelial SRA01/04 cells were induced by high glucose (HG) and subsequently treated with Pae. Cell viability was detected using the MTT assay. Moreover, LDH levels were detected. Immunofluorescence (IF) and Western blotting were used to determine the protein expression levels of N-cadherin and E-cadherin. ELISA was performed to determine oxidative stress-related indicator levels. TUNEL and Western blotting detected the apoptotic rate. The mRNA and protein expression levels of sirtuin 1 (SIRT1) in SRA01/04 cells were measured via reverse transcription-quantitative PCR and Western blotting, respectively. Subsequently, cell transfection techniques were used to inhibit the expression of SIRT1 in cells. MTT, ELISA, IF, Western blotting and TUNEL assays were used to investigate the mechanisms of epithelial-mesenchymal transition (EMT) and oxidative damage with Pae in the diabetic cataract. Pae significantly increased cell viability and possibly inhibit the EMT and oxidative damage of SRA01/04 cells induced by HG. Pae was demonstrated to upregulate SIRT1 expression levels. The results therefore suggested that the downregulation of SIRT1 reversed the protective effect of Pae on EMT and oxidative damage in SRA01/04 cells induced by HG. In conclusion, Pae may inhibit EMT of lens epithelial cells and reduce oxidative damage in diabetic cataracts via the upregulation of SIRT1.

Drug-eluting intraocular lens with sustained bromfenac release for conquering posterior capsular opacification

Cataract is the leading cause of visual impairment, and posterior capsular opacification (PCO) is the most common long-term complication of modern cataract surgery, which can cause severe visual impairment after surgery. The proliferation, migration, and epithelial-mesenchymal transition (EMT) of residual lens epithelial cells (LECs) stimulated by growth factors and cytokines, are the key pathological mechanisms involved in the development of PCO. This study demonstrated that non-steroidal anti-inflammatory drug (NSAID), bromfenac, was capable of effectively inhibiting cell migration, overexpression of EMT markers, such as fibronectin (FN), matrix metalloproteinase 2 (MMP2), α-smooth muscle actin (α-SMA), and transcription factor Snail, and extracellular signal-regulated kinase (ERK)/glycogen synthase kinase-3β (GSK-3β) signaling induced by transforming growth factor-β2 (TGF-β2) in vitro. The inhibitory effect of bromfenac on TGF-β2-induced EMT was also verified on a primary lens epithelial cell model using human anterior capsules. Furthermore, based on ultrasonic spray technology, we developed a drug-eluting intraocular lens (IOL) using poly (lactic-co-glycolic acid) (PLGA) with sustained bromfenac release ability for the prevention of PCO development. In the rabbit models of cataract surgery, bromfenac-eluting IOL exhibited remarkable PCO prevention and inflammation suppression effects with excellent biocompatibility. In conclusion, bromfenac can inhibit TGF-β2-induced cell migration and the EMT of LECs via ERK/GSK-3β/Snail signaling. The present study offers a novel approach for preventing PCO through PLGA-based drug sustained-release IOLs.

LncRNA-MALAT1/miRNA-204-5p/Smad4 Axis Regulates Epithelial–Mesenchymal Transition, Proliferation and Migration of Lens Epithelial Cells

ABSTRACT Purpose: Posterior capsular opacification (PCO), a common complication after cataract surgery, primarily originated from the epithelial–mesenchymal transition (EMT), proliferation, and migration of human lens epithelial cells (LECs). This study aimed to explore whether the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/miR-204-5p/Smad4 axis are involved in EMT of LECs. Materials and methods LECs were cultured and induced with TGF-β2 (10 ng/mL). SiRNA against MALAT1 (Si‐MALAT1) was transfected into LECs to knockdown the expression of MALAT1. To overexpress or knockdown miR‐204-5p, miR-204-5p mimics (miR-204-5p mimics) and anti-miR-204-5p (miR-204-5p inhibitor) were transfected into LECs. We used RNA FISH to identify the location of MALAT1. RNA levels of MALAT1 and miR-204-5p were analyzed by RT-qPCR. Additionally, target protein levels of Smad4, epithelial differentiation and mesenchymal markers were analyzed with Western blot. We employed EdU Labeling to measured cell proliferation and performed Transwell Assay to analyze the cell migration. Dual-luciferase reporter assays in LECs were conducted to verify whether miRNA-204-5p was negatively regulated by MALAT1 and Smad4 was a direct target of miR-204-5p. Results The expression of MALAT1 was upregulated in PCO specimens. MALAT1 was overexpressed in TGF-β2 induced LECs, and the knockdown of MALAT1 could attenuate TGF-β2 induced EMT. Besides, the upregulation of MALAT1 was correlated with the downregulation of miR-204-5p and upregulation of Smad4. Importantly, MALAT1 was revealed to be located in the cytoplasm of LECs. Furthermore, luciferase reporter assays confirmed that MALAT1 could negatively regulate the expression of miR-204-5p and then regulate its direct target Smad4. Finally, the knockdown of MALAT1 could inhibit the EMT, proliferation, and migration of LECs; however, those can be reversed by anti-miR-204-5p. Conclusions Our findings reveal that MALAT1 may regulate EMT, proliferation, and migration of LECs as a ceRNA by “sponging” miR-204-5p and targeting Smad4, and serve as a promising therapeutic target in preventing PCO.

Autophagy inhibition attenuates TGF-β2-induced epithelial–mesenchymal transition in lens epithelial cells

AimsAutophagy has been reported to play an essential role in fibrotic disorders. Known as fibrotic cataract, posterior capsular opacification (PCO) result from pathological epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). This study aims to identify the role and potential mechanism of autophagy in TGF-β2-induced EMT in LECs. Main methodsPrimary rabbit LECs were treated with TGF-β2 to induce EMT as a model of fibrotic cataract in vitro. 3-methyladenine, chloroquine, bafilomycin A1, and gene silencing of autophagy-related protein 7 (ATG7) were treated in LECs for autophagy inhibition, while rapamycin was utilized for autophagy activation. The expression levels of EMT/autophagy-associated markers were analyzed by qRT-PCR, western blotting, immunofluorescence and transmission electron microscopy. We additionally examined cell migration ability with transwell migration assay and wound healing assay. Key findingsTGF-β2 promoted autophagy flux during EMT progression of LECs in a time-dependent manner. Autophagy activation by rapamycin enhanced TGF-β2-triggered fibrogenic responses and cell migration in LECs, whereas pharmacological inhibition of autophagy alleviated TGF-β2-induced increases of EMT markers and cell migration of LECs. In addition, the phosphorylation of Smad2/3 induced by TGF-β2 was suppressed through autophagy inhibition, while it was promoted upon autophagy activation, indicating that TGF-β2/Smad signaling was involved in the modulation of autophagy on EMT in LECs. Furthermore, ATG7-silenced LECs exerted anti-fibrosis effect induced by TGF-β2 through downregulation of autophagy. SignificanceIntervention/inhibition of autophagy could attenuate TGF-β2-induced EMT in LECs, which provides autophagy-related insights on preventing and treating the fibrotic cataract or other fibrotic diseases.

MicroRNA-184 is a key molecule responsible for the transforming growth factor-β2 -induced epithelial-mesenchymal transition in human lens epithelial-B3 cells.

TGF-β2-induced epithelial-mesenchymal transition (EMT) is an important mechanism for posterior capsule opacity (PCO) in lens epithelial cells (LECs). This study aimed to investigate if MicroRNA-184 (miR-184) plays a role in the TGF-β2-induced EMT in LECs. Human LECs (HLE-B3 cells) were used in this study. Quantitative real-time polymerase chain reaction (PCR) (qRT-PCR) was performed to analyse miR-184 expressions in HLE-B3 treated with TGF-β2 at different concentrations (0-15 ng/mL) and different time (10 ng/mL, 0-48 hours). After transfection of miR-184 mimics or miR-184 inhibitor, cells were treated with 10 ng/mL TGF-β2 for 24 hours, and the expression levels of miR-184, E-cadherin, vimentin, zinc finger E-box binding homeobox 2 (ZEB2), α-Smooth muscle actin (α-SMA), Collagen 1 and bin3 were determined by qRT-PCR and Western blot, respectively. TGF-β2 treatment significantly downregulated E-cadherin and upregulated vimentin generally in a dose-dependent and time-dependent manner. TGF-β2 treatment significantly elevated the level of miR-184 in both dose- and time-dependent manners. In addition, transfection of miR-184 inhibitor RNA significantly attenuated TGF-β2-induced downregulation of E-cadherin as well as upregulation of vimentin, ZEB2, α-SMA and Collagen 1, whereas transfection of miR-184 mimic further enhanced the effects of TGF-β2 on the expressions of these markers. Furthermore, TGF-β2 treatment significantly downregulated bin3, and transfection of miR-184 mimic and miR-184 inhibitor significantly enhanced and attenuated the inhibition effect of TGF-β2 on bin3, respectively. miR-184 plays a key role in the TGF-β2-induced EMT in LECs, and bin3 may be a downstream protein.

A HGF‑derived peptide suppresses EMT in human lens epithelial cells via the TGF‑β/Smad and Akt/mTOR signaling pathways.

Posterior capsule opacification(PCO) as a result of proliferation and fibrogenesis of lens epithelial cells(LECs) is the most frequent long‑term complication of modern cataract surgery. LECs may undergo epithelial‑mesenchymal transition(EMT) that resembles the morphological and molecular characteristics of PCO. A pre‑identified novel, hepatocyte growth factor(HGF)‑derived peptide H‑RN, was reported to exhibit anti‑angiogenic activity and anti‑inflammatory effects in ocular cells both invitro and invivo. However, the role of H‑RN in the promotion of the development of EMT in LECs is unknown. In the present study, the effects of H‑RN on the development of EMT induced by transforming growth factor(TGF)‑β in human LECs, and the possible signaling pathways participating in this process were investigated. The results showed that H‑RN promoted the expression of the EMT‑associated markers, α‑smooth muscle actin and fibronectin, whereas the expression of E‑cadherin and connexin43 were reduced. The morphological changes typically associated with EMT seen in LECs induced by TGF‑β2 were inhibited by H‑RN, which was consistent with the effects of a TGF‑β2 inhibitor, SB431542. Smad2 and Smad3 phosphorylation induced by TGF‑β2 were reduced by H‑RN, and phosphorylation of Akt, mTOR and P70S6K induced by TGF‑β2 were also notably reduced by H‑RN in LECs. Therefore, the results of the present study showed that H‑RN treatment significantly suppressed the development of EMT induced by TGF‑β2, at least partially through the TGF‑β/Smad and Akt/mTOR signaling pathways in human LECs. The present study highlights that H‑RN, a novel HGF‑derived peptide, may be a novel therapeutic agent for prevention and treatment of PCO.

Let-7a-5p represses proliferation, migration, invasion and epithelial-mesenchymal transition by targeting Smad2 in TGF-β2-induced human lens epithelial cells

Transforming growth factor β2 (TGF-β2)/Smad signaling is widely accepted as a key inducer of proliferation and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs), contributing to the development of posterior capsule opacification (PCO). Increasing evidence shows that microRNAs (miRNAs) play important roles in PCO pathogenesis. Herein, we aimed to explore the role and molecular mechanism of let-7a-5p on TGF-β2-induced proliferation and EMT in LECs. qRT-PCR was performed to detect the expression of let-7a-5p and Smad2 mRNA. Western blot was used to determine the Smad2 level and the induction of EMT. The targeted correlation between let-7a-5p and Smad2 was confirmed using dual-luciferase reporter and RNA immunoprecipitation assays. CCK-8 assay was employed to determine cell proliferation, and transwell assays were performed to assess cell migration and invasion. We found that TGF-β2 induced EMT of LECs, and TGF-β2 upregulated Smad2 expression and reduced let-7a-5p expression in LECs. Smad2 was a direct target of let-7a-5p. Moreover, let-7a-5p upregulation repressed proliferation, migration, invasion and EMT in TGF-β2-induced LECs. But, Smad2 expression restoration abrogated the inhibitory effect of let-7a-5p upregulation. In conclusion, our data indicated that let-7a-5p upregulation repressed TGF-β2-induced proliferation, migration, invasion and EMT at least partly by targeting Smad2 in LECs, highlighting that let-7a-5p might act as a promising therapeutic target to intervene to the progression of PCO.

Curcumin Inhibits Proliferation and Epithelial-Mesenchymal Transition in Lens Epithelial Cells through Multiple Pathways.

Background Posterior capsule opacification (PCO), a complication of extracapsular lens extraction surgery that causes visual impairment, is characterized by aberrant proliferation and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs). Curcumin, exerting inhibitive effects on cell proliferation and EMT in cancer, serves as a possible antidote towards PCO. Methods Cellular proliferation of LECs after treatment of curcumin was measured with MTT assay and flow cytometry. The transcriptional and expressional levels of proteins related to proliferation and EMT of LECs were quantified by western blotting and real-time PCR. Results Curcumin was found to suppress the proliferation of LECs by inducing G2/M arrest via possible inhibition of cell cycle-related proteins including CDK1, cyclin B1, and CDC25C. It had also inactivated proliferation pathways involving ERK1/2 and Akt pathways in LECs. On the other hand, curcumin downregulated the EMT of LECs through blocking the TGF-β/Smad pathway and interfering Notch pathway which play important roles in PCO. Conclusions This study shows that curcumin could suppress the proliferation and EMT in LECs, and it might be a potential therapeutic protection against visual loss induced by PCO.

2',3'‐Cyclic‐nucleotide 3'‐phosphodiesterase contributes to epithelial‐mesenchymal transition of lens epithelial cells through the notch signalling pathway

ObjectivesFibrosis is a complex process involved in multiple diseases that result in organ injury and failure. Cataract, one common form of ocular fibrosis, is a main cause of blindness worldwide, and surgery may be the only cure. In this regard, epithelial‐mesenchymal transition (EMT) of lens epithelial cells (LECs) is the primary cause of anterior subcapsular cataract (ASC). This study aimed to investigate the mechanism by which 2',3'‐cyclic‐nucleotide 3'‐phosphodiesterase (CNPase) regulates the function of EMT in LECs.Materials and MethodsA mouse model of ASC was used to observe the expression of CNPase in the lens and correlate its expression changes with lens EMT. Furthermore, the effects of CNPase on cell migration and cell proliferation were evaluated by transwell migration, wound healing and EdU staining assays. Finally, Western blotting and immunofluorescence were used to assess the mechanical properties potentially involved in the regulation of EMT by CNPase.ResultsThe expression of CNPase was upregulated in LECs during the EMT process in mice with ASC. Notably, CNPase significantly promoted the proliferation, migration and EMT of LECs in vitro. Interestingly, the EMT‐promoting mechanism of CNPase may be achieved by targeting the Notch signalling pathway.ConclusionsConsidering the involvement of EMT in ASC, both CNPase and the Notch signalling pathway may be therapeutic targets for the treatment of cataracts.